Stereoisomer separation method using antibody combing site-containing molecules

ABSTRACT

A method of separating a pair of stereoisomers, and especially enantiomers, using monoclonal receptor molecules containing an antibody combining site that binds to substantially only one of that pair of stereoisomers is disclosed.

This invention was made with government support under Contract GM 35318awarded by the National Institute of Health and Contract DCB 8607352issued by the National Science Foundation. The government has certainrights in the invention.

This is a division of application Ser. No. 07/083,681, filed Aug. 1,1987, now U.S. Pat. No. 5,079,152, which was a continuation-in-part ofapplication Ser. No. 07/055,177, filed May 28, 1987, now U.S. Pat. No.4,900,674.

TECHNICAL FIELD

The present invention relates to antibodies (receptors), antigens andimmunogens (ligands), and more particularly to receptor molecules thatcontain an antibody combining site that stereospecifically binds andthereby stabilizes a transition state leading to a stereospecificproduct and exhibits catalytic properties, as well as to use of suchreceptors to separate stereoisomers.

BACKGROUND OF THE INVENTION

Binding phenomena between ligands and receptors play many crucial rolesin biological systems. Exemplary of such phenomena are the binding ofoxygen molecules to deoxyhemoglobin to form oxyhemoglobin, and thebinding of a substrate to an enzyme that acts upon it such as between aprotein and a protease like trypsin. Still further examples ofbiological binding phenomena include the binding of an antigen to anantibody, and the binding of complement component C3 to the so-calledCR1 receptor.

Many drugs and other therapeutic agents are also believed to bedependent upon binding phenomena. For example, opiates such as morphineare reported to bind to specific receptors in the brain. Opiate agonistsand antagonists are reported to compete with drugs like morphine forthose binding sites.

Ligands such as man-made drugs, like morphine and its derivatives, andthose that are naturally present in biological systems such asendorphins and hormones bind to receptors that are naturally present inbiological systems, and will be treated together herein. Such bindingcan lead to a number of the phenomena of biology, including particularlythe hydrolysis of amide and ester bonds as where proteins are hydrolyzedinto constituent polypeptides by an enzyme such as trypsin or papain, orwhere a fat is cleaved into glycerine and three carboxylic acids,respectively. In addition, such binding can lead to formation of amideand ester bonds in the formation of proteins and fats, as well as to theformation of carbon to carbon bonds and carbon to nitrogen bonds.

Immunological binding can be used to experimentally divert bindinginteractions to catalytic processes. Jencks, W. P., Catalysis inChemistry and Enzymology, page 288 (McGraw-Hill, New York 1969).Attempts to introduce reactive groups into a combining site of anantibody, however, have generally been unsuccessful. Royer, G. P., Adv.Catal., 29, 197 (1980). Some monoclonal antibodies are reported toinclude nucleophilic residues that react with an activated ester,appendage on a homologous hapten recognized by the antibody. Kohen etal., FEBS Lett., 111, 427 (1980); Kohen et al., Biochem. Biophys. Acta,629, 328 (1980) and Kohen et al., FEBS Lett., 100, 137 (1979). In thesecases, the rate of acylation of the nucleophile is presumablyaccelerated by its proximity to a binding site of the haptenic fragment.

These constructs, though interesting, are severely limited by theirfailures to address the mechanism of binding energy utilization that isessential to enzymes [W. P. Jencks, Adv. Enzymol., 43, 219 (1975)].Aside from that failure, when strong binding is directed to stablestates, the slow rate of dissociation of the complex can impedecatalysis.

The above deficiencies can be redressed by using a transition stateanalog as the hapten to elicit the desired antibodies. This hapten (alsoreferred to herein as an "analog-ligand") can assume the role of aninhibitor in the catalytic system.

Hydrolysis of amide and ester bonds is thought by presently acceptedchemical theory to proceed in aqueous media by a reaction at thecarbonyl carbon atom to form a transition state that contains atetrahedral carbon atom bonded to (a) a carbon atom of the acid portionof the amide or ester, (b) two oxygen atoms, one being from the carbonylgroup and the other from a hydroxyl ion or water molecule of the medium,and (c) the oxygen atom of the alcohol portion of an ester or thenitrogen atom of the amine portion of an amide. Transition states ofsuch reactions are useful, well-accepted mental constructs that bydefinition, cannot be isolated, as compared to intermediates, which canbe isolated.

Although the above hydrolytic and other transition states can not beisolated, a large amount of scientific literature has been devoted tothe subject. Some of that literature is discussed hereinafter.

Whereas the before-described transition state for amide and esterhydrolyses is believed to be well understood, the parameters of thetopology, e.g., size, shape (stereoconfiguration) and charge, ofreceptor binding sites in which particular amides, such as proteins, oresters, such as fats, react through those transition states is not aswell understood. It would therefore be beneficial if the topology of aplurality of binding sites were known so that the interactions of theligands that bind in those sites could be studied. Unfortunately, thetopology of receptor binding sites in biological hydrolyses is generallyunknown, except for a relatively small number of enzymes whose X-raycrystal structures have been determined.

This lack of knowledge of binding site topology stems in part from alack of knowledge of even the location in cells of many binding sites ofreceptors. In addition, for those receptor binding sites whose locationsare known, the chemical identity; i.e., protein and carbohydratecomposition, of the binding site is generally unknown. Thus, theinvestigator is generally stymied in seeking to understand thetopological requirements of receptor binding sites and therefore inseeking to construct therapeutic agents that can fulfill thoserequirements.

Investigators must therefore screen potential therapeutic agents inanimal or cell culture studies to ascertain whether a potentialtherapeutic agent may be useful. Such systems, while useful, areexpensive and time-consuming to use.

Even where the topology and chemical reactivity of a hydrolytic receptorsuch as an enzyme are known, enzymes such as hydrolytic proteasestypically cleave their substrates, polypeptide chains, adjacent to aparticular amino acid residue that may occur several times in thepolypeptide chain of the protein. Although such relatively randomcleavage can be useful in obtaining a polypeptide map of the protein,that relatively random cleavage is not as useful where particular aminoacid residue sequences are desired to be produced.

Recently, Lerner, Tramontano and Janda [Science, 234, 1566 (1986)]reported monoclonal antibodies that catalytically hydrolyzed an ester.Tramontano and Lerner, also describe using monoclonal antibodies tohydrolyze esters in U.S. Pat. No. 4,656,567. Pollack, Jacobs and Schultz[Science, 234, 1570 (1986)] reported a myeloma protein denominatedMOPC167 [Leon et al., Biochem., 10, 1424 (1971)] that catalyzes thehydrolysis of a carbonate.

In the two Lerner and Tramontano disclosures, the antibodies were raisedto a phosphonate that was synthesized to represent a stable analog ofthe tetrahedral hydrolytic transition state of the carboxylic acid esteror amide. The Pollack et al. antibody principally discussed was amyeloma protein that happened to bind to a phosphonate that wasstructurally analogous to the carbonate analog hydrolyzed. Thus, in theLerner and Tramontano et al. work, the substrate to be hydrolyzed waspreselected, with the immunizing analog and hydrolytic antibodies beingsynthesized in accordance with the desired product. Pollack et al.designed the substrate to be hydrolyzed once they knew the specificityof the myeloma protein. Pollack et al. also reported (above) theexistence of a catalytic antibody, substrate and analog substrate systemfor carbonate hydrolysis similar in concept to that of Lerner et al.Work relating to that system is reported in Jacobs et al., J. Am. ChemSoc., 109:2174 (1987).

Published patent application WO 85/02414 discusses the possible use ofantibodies as catalysts, and presents data relating to the use ofpolyclonal serum in hydrolyzing o-nitrophenyl-beta-D-galactoside. Theantibodies useful in that application are said to be inducible by areactant, a reaction intermediate or to an analog of the reactant,product or reaction intermediate. The term "analog" is there defined toencompass isomers, homologs or other compounds sufficiently resemblingthe reactant in terms of chemical structure that an antibody raised toan analog can participate in an immunological reaction with the reactantbut will not necessarily catalyze a reaction of the analog.

The data provided in that specification only indicate that some cleavageof the substrate (reactant) galactoside occurred over an eighteen hourtime period using a relatively concentrated antibody preparation (1:10and 1:20 dilutions). Although catalysis was alleged, catalytic activitywas not shown since no turn over of the allegedly catalytic antibody wasshown, nor was there an indication of the percentage of substrategalactoside cleaved. That application did indicate thatbeta-D-galactosidase cleaved about ten times as much substrate as didthe polyclonal antibodies, presuming linearity of absorbance at theunnamed concentration of substrate studied.

From the data presented in that application, it is possible that anucleophilic replacement of the o-nitrophenyl group occurred by aterminal amino group of a lysine residue of the antibody preparationused. Thus, the observed absorbance could have been due to formation ofepsilon-amino lysinyl o-nitrophenyl aniline or to the formation of anepsilon-amino-lysinyl galactoside and o-nitrophenol, either of whichoccurrences would not be catalytic since the antibody was consumed,rather than turning over.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a catalytic molecule. That moleculecontains an antibody combining site that itself constitutes thecatalytically active portion of the molecule. An antibody combining sitecan also be referred to as a paratope or an idiotype-containingpolyamide. The catalytic molecule is generally referred to herein as areceptor molecule, or more simply, a receptor.

A receptor molecule of the present invention is preferably monoclonal(discussed hereinafter), and contains an antibody combining site that iscapable of catalyzing the formation of a preselected bond such as acarboxylic amide or ester bond, preferably a lactone bond.

In enzymology, a protein that catalyzes a bond-forming reaction can bereferred to as a synthase. A protein that catalyzes an amide- orester-forming reaction can be said to be a member of the amide or estersynthase family of enzymes. Using that nomenclature system for thepreferred formation of a lactone, a lactone-forming enzyme can bereferred to as a lactone synthase. The receptors of this invention willbe described herein as exhibiting synthase activity when discussedbroadly, amide or ester synthase activity when discussed at intermediatebreadth, and will be described as exhibiting lactone synthase activitywhen discussed in relation to the specific reactions described herein asexemplary.

The synthesis reactions discussed herein are preferably stereospecific.By stereospecific (or stereoselective) it is meant that one of at leasttwo stereoisomers is formed preferentially in the reaction.

Stereoisomers can be geometric isomers such as cis and trans isomers oroptical isomers that are also called enantiomers. Geometric andenantiomeric isomers can also exist in the same molecule, particularlymolecules containing rings, and such stereoisomers are alsocontemplated.

A receptor capable of catalyzing stereoselective synthesis iscontemplated herein. Such a stereoselective synthase molecule ispreferably monoclonal and catalyzes the synthesis of a preselectedproduct that contains relatively more of one stereoisomer than the otherstereoisomer.

The stereoselective receptor synthase molecule contains an antibodycombining site capable of catalyzing the stereoselective synthesis of adesired product. That antibody combining site binds to substantiallyonly one stereoisomer (geometric or optical) of a reactant ligand thatis structurally capable of forming the product, and also binds tosubstantially only one stereoisomer of a ligand structurally analogous(analog-ligand) to a transition state leading to one stereoisomer of theproduct. Thus, for example, the receptor binds selectively to oneenantiomer of the analog-ligand, and to one enantiomer of the reactantligand to preferentially form one enantiomer of the product, whereenantiomers are the stereoisomers of interest.

The present invention also contemplates a molecule exhibiting amide orester synthase activity that comprises a receptor molecule. Thepreferably monoclonal receptor contains an antibody combining sitecapable of catalyzing the formation of a preselected carboxylic amide orester bond. The combining site binds to: (a) a reactant ligandcontaining a carbonyl group carbon atom and an amine or alcohol groupthat is structurally capable of forming the preselected carboxylic amideor ester bond (the reactant ligand); and (b) a ligand structurallyanalogous to the preselected amide or ester; the analog-ligand having atetrahedrally bonded phosphorus atom located at the position occupied bythe above-mentioned carbonyl carbon atom of the preselected carboxylicamide or ester bond of the before-mentioned ligand. The tetrahedrallybonded phosphorus atom is itself directly bonded to: (i) thealpha-carbon atom of the acid portion of the analog ligand by a singlebond; (ii) a first oxygen atom that is doubly bonded to the phosphorusatom; (iii) a second oxygen atom that is bonded to the phosphorus atomby a single bond, and is singly bonded to a radical selected from thegroup consisting of hydrogen, C₁ -C₆ lower alkyl, benzyl and phenyl; and(iv) a third oxygen atom or a nitrogen atom singly bonded to thephosphorus atom, and is also singly bonded to the alpha-carbon atom ofthe amine or alcohol portion of the analog ligand.

A receptor capable of catalyzing the formation of an excess of apreselected enantiomeric carboxylic acid amide or ester product such asa lactam or a lactone over the other enantiomer is further contemplated.Here, the receptor amide or ester synthase contains an antibodycombining site capable of catalyzing the formation of a preselectedenantiomer of the amide or ester product, and the combining site bindsto: (a) substantially only one of a ligand enantiomeric pair thatcontains a carbonyl group carbon atom and an amine or alcohol groupstructurally capable of forming the preselected amide or ester (reactantligand enantiomer), and (b) a ligand structurally analogous(analog-ligand) to one enantiomer of a transition state leading to thepreselected amide or ester product.

The analog-ligand contains a tetrahedrally bonded phosphorus atomlocated at the position occupied by the carbon atom of the carbonylgroup of the preselected carboxylic amide or ester product. Thattetrahedrally bonded phosphorus atom being bonded directly to: (i) thealpha-carbon atom of the acid portion of the analog-ligand by a singlebond; (ii) a first oxygen atom that is doubly bonded to the phosphorus;(iii) a second oxygen atom that is bonded to said phosphorus atom by asingle bond, and is singly bonded to a radical selected from the groupconsisting of hydrogen, C₁ -C₆ lower alkyl, benzyl and phenyl; and (iv)a third oxygen atom or a nitrogen atom that is singly bonded to thephosphorus atom, and is also singly bonded to the alpha-carbon atom ofthe alcohol or amine portion of the analog-ligand.

In preferred practice, the receptor molecule exhibits stereoselectivelactam or lactone synthase activity. A particularly preferred synthasemolecule is the monoclonal antibody denominated 24B11. Thus, thereactant ligand containing the carbonyl carbon is capable of forming alactone, and the analog ligand is a cyclic phosphonate. In particularlypreferred practice, the ligand and analog-ligands have structures offormulas I and II, respectively, below: ##STR1##

wherein R is hydrogen, C₁ -C₆ lower alkyl or a linking group bonded toan antigenic carrier; and R² is hydrogen or C₁ -C₆ lower alkyl, and thereceptor preferentially immunoreacts with one enantiomer of the ligandand one enantiomer of the analog-ligand.

A method for carrying out a stereoselective synthesis to prepare apreselected product that contains relatively more of one stereoisomerthan another stereoisomer is contemplated as another aspect of theinvention. For this synthesis, a reactant ligand and an effective amountof a synthase molecule are admixed in an aqueous medium to form areaction medium; the reactant ligand being structurally capable offorming two stereoisomers of the desired product. The synthase moleculecomprises a receptor molecule, preferably monoclonal, that contains anantibody combining site capable of stereospecifically catalyzing theformation of the product. That antibody combining site binds to: (a)substantially only one stereoisomer of the ligand, and (b) a ligandstructurally analogous to a transition state leading to one stereoisomerof the product (analog-ligand).

The reaction mixture is maintained for a period of time sufficient forthe stereoisomeric product to form. The product, containing relativelymore of one stereoisomer; i.e., the stereoisomer whose formation wascatalyzed by the synthase molecule, than another stereoisomer isthereafter typically recovered.

Another embodiment contemplates a method for forming (synthesizing) apreselected enantiomeric carboxylic acid amide or ester product thatcontains an excess of one enantiomer over the other enantiomer. Here, anenantiomeric reactant ligand pair structurally capable of forming theenantiomeric carboxylic amide or ester is admixed with an effectiveamount of an amide or ester synthase molecule to form a reactionmixture.

The reactant ligand contains a carbonyl group carbon atom and an amineor alcohol group capable of forming the preselected carboxylic acidamide or ester product. The amide or ester synthase molecule comprises areceptor molecule, preferably monoclonal, that contains an antibodycombining site capable of catalyzing the formation of the preselectedenantiomer of the amide or ester product.

That combining site binds to: (a) substantially only one enantiomer ofthe enantiomeric reactant ligand pair, and (b) a ligand structurallyanalogous to one enantiomer of a transition state leading to thepreselected amide or ester product. The enantiomer of the analog-ligandthat is bound by that combining site has a tetrahedrally bondedphosphorus atom located at the position occupied by the carbon atom ofthe carbonyl group of the carboxylic acid amide or ester product.

The tetrahedrally bonded phosphorus atom of the analog ligand is bondeddirectly to: (i) the alpha-carbon atom of the acid portion of theanalog-ligand by a single bond; (ii) a first oxygen atom that is doublybonded to the phosphorus atom; (iii) a second oxygen atom that is singlybonded to the phosphorus atom, and is singly bonded to a radicalselected from the group consisting of hydrogen, C₁ -C₆ lower alkyl,benzyl and phenyl; and (iv) a third oxygen atom or a nitrogen atom thatis singly bonded to the phosphorus atom, and is also singly bonded tothe alpha-carbon atom of the amine or alcohol portion of theanalog-ligand.

The reaction mixture is maintained for a time period sufficient for thepreselected amide or ester product enantiomer to form. The product isthereafter again typically recovered containing an excess of oneenantiomer over the other.

A method of forming a preselected carboxylic acid amide or esterconstitutes still another aspect of the present invention. Here, aligand containing a carbonyl group carbon atom and an amine or alcoholgroup structurally capable of forming the preselected carboxylic acidamide or ester is admixed in an aqueous medium with an effective amountof an amide or ester synthase molecule to form a reaction mixture. Here,however, the reactant ligand and analog-ligand need not havestereoisomers, nor need the product have stereoisomers. The reactionmixture is thereafter maintained for a time period sufficient for theamide or ester to form; and the product is thereafter preferablyrecovered.

The invention contemplates a reactant ligand structurally capable offorming an amide or ester as well as an analog-ligand containing ananalog to the amide- or ester-forming transition state leading to theamide or ester product. Those molecules differ in the fact the ligandcontains a carbonyl group and an amine or alcohol capable of forming anamide or ester, whereas the analog-ligand contains a phosphorus centralatom in a structure that mimics the amide- or ester-forming transitionstate. The ligand and analog-ligand can also differ in the substitutionof the atoms bonded to the central atom at a position at least one atomaway from the central atom since the analog-ligand must possesssufficient stability to be used as an immunizing hapten, and be bound toan antigenic carrier.

In the studies described herein, phosphonate monoaryl esters function astransition state analogs to generate antibodies that exhibit amide orester synthase activity, and specifically, stereoselective lactonesynthase activity. In effect, these antibodies express their inherentbinding energy functionally, as true enzymes, to form an amide (lactam)or ester (lactone), and classically, as antibodies, to bind antigens.

An exemplary immunizing analog-ligand molecule that constitutes ananalog of a lactone-forming transition state is represented by acompound having a structure of formula III: ##STR2##

wherein X is O or NH;

R is hydrogen, C₁ -C₆ lower alkyl, or a linking group alone or bonded toan antigenic carrier; and

R¹ is hydrogen, C₁ -C₆ lower alkyl, benzyl or phenyl.

The analog-ligand transition state molecules contemplated in thisinvention are of relatively small molecular size, as compared to aprotein, and are therefore typically linked to a larger, antigeniccarrier molecule when used to induce the production of a receptormolecule. Such relatively small molecules are commonly referred to ashaptens. These analog-ligand molecules also typically contain a linkingatom or group such, as a reactive mercaptan, a succinimide or activatedcarbonylic acid ester group that provides a means to attach the haptenicanalog-ligand molecules to an antigenic carrier for use as an immunogen,as shown in the formula above.

A method of preparing a synthase molecule such as an amide or estersynthase is also contemplated. Here, a before-described haptenicanalog-ligand molecule that comprises a transition state analog of theproduct to be formed such as an amide- or ester-forming transition stateanalog is provided linked to an antigenic carrier as an immunogenicconjugate. The conjugate thus provided is dissolved or dispersed in aphysiologically tolerable diluent to form an inoculum. The inoculum isintroduced as by injection into a mammalian host such as a laboratoryanimal or a horse in an amount sufficient to induce antibodies to thehaptenic analog-ligand. The antibodies so induced are harvested. Theharvested antibodies that immunoreact with the immunizing, haptenicanalog-ligand and catalyze the desired reaction are then collected.

In particularly preferred practice, monoclonal antibodies are prepared.Here, the above immunizing technique is used and the harvestedantibodies are assayed for their ability to bind to (immunoreact with)the immunizing, haptenic ligand analog. Immunoglobulin-producing cellssuch as those from the spleen of an animal whose antibodies bind to theimmunizing, haptenic analog-ligand are collected and are fused withmyeloma cells to form hybridoma cells. The hybridoma cells are grown ina culture medium and the supernatant medium from the growing hybridomacells is assayed for the presence of antibodies that bind to theimmunizing, haptenic analog-ligand and catalyze the desired reaction ofthe reactant ligand. Hybridoma cells whose supernatant contains suchbinding, catalytic antibodies are then cloned to provide the desiredmonoclonal antibodies from culture medium supernatant or from theascites of another host mammal into which the hybridoma is introduced.

Where the analog-ligand is capable of exhibiting stereoisomerism, themonoclonal antibodies selected preferably immunoreact stereospecificallywith one of those stereoisomers in preference to another stereoisomer.For example, the analog-ligand whose structure is depicted in formulaIII has a chiral carbon atom at the position at which the side chainjoins the ring and therefore exists as an enantiomeric pair, unless theracemic modification is resolved. A particularly preferred lactonesynthase of this invention, the monoclonal antibody secreted byhybridoma 24B11, stereoselectively immunoreacts with one of thoseenantiomers as compared to the other, as is the case for thecorresponding reactant ligand, and thereby preferentially catalyzesformation of one enantiomeric product over the other enanitomer.

The described polyclonal or monoclonal antibodies can be used as thesynthase molecules of this invention. Alternatively, the so-called Fc orFc' portions of the antibodies can be removed as by enzymic cleavage toprovide an antibody combining site (paratope or idiotype-containingpolyamide) that binds to the immunizing, haptenic analog-ligand such asFab or F(ab')₂ antibody portion, respectively.

The polyclonal, monoclonal and idiotype-containing polyamide receptorsalso bind to the reactant ligand capable of forming the desired productsuch as an amide or ester. Such binding typically leads to catalyzedformation of the preselected bond, e.g., the amide or ester bond.

Still another embodiment of the present invention is a method forseparating one of a pair of stereoisomers from the other. In thisaspect, a receptor molecule, preferably monoclonal, is admixed with apair of stereoisomers in an aqueous medium to form an admixture. Thisreceptor contains an antibody combining site that immunoreactsstereoselectively with substantially only one of the stereoisomers. Theadmixture so formed is maintained for a time period sufficient for thereceptor to immunoreact with (bind to) one of the stereoisomers to forman immunoreactant within the admixture. The immunoreactant is thereafterseparated from the remaining admixture, thereby separating one of thestereoisomers from the other. The bound stereoisomer can be separatedfrom the receptor of the immunoreactant in a further step.

The present invention provides several benefits and advantages. Onebenefit is the preparation of receptors whose binding site topologicalrequirements are tailored to a particular ligand to be studied.

Another benefit of the present invention is the preparation of receptorsthat form an amide or ester bond at a predetermined site and thatexhibit catalytic properties.

An advantage of the invention is that because of the specificity of thereceptors that are produced, a reactant ligand containing a plurality ofdifferent reactive groups capable of forming bonds can be caused to formthe desired bond at a preselected, particular site.

Another advantage of the present invention is that it provides a meansfor stereoselective syntheses.

Still another benefit of the invention is that one enantiomeric productcan be prepared in excess over the other enantiomer.

Still another advantage of the invention is that is provides a methodfor separating stereoisomers such as enantiomers that is far easier thanpreviously known techniques.

Still further benefits and advantages of the present invention will beapparent to those skilled in the art from the disclosures that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which constitute a portion of this disclosure:

FIG. 1 is a schematic representation showing a reactant ligandstructurally capable of forming a lactone (on the left), a proposedlactone-forming transition state for the ligand (bracketed, uppercenter), a structure of the analog-ligand (bracketed, lower center), andthe lactone product of the reaction (on the right). Thestereoconfigurations of one ligand enantiomer, the transition state,analog-ligand, and product are illustrated to show one possiblestereochemical pathway. In that stereochemical view, solid lines are inthe plane of the page, bonds projecting outwardly from the page areshown by solid triangular bonds, bonds projecting backwardly from thepage are shown by a dashes, and a bond being formed is shown as a dottedline. The symbols δ⁻ and δ⁺ are utilized to show relative negative andpositive charges, and the symbol designates the transition state.

FIG. 2 is graph showing the decrease of reactant ligand capable offorming a lactone shown in FIG. 1 (substrate) in reaction media overtime in minutes (min.) in the presence of (i) monoclonal antibody (Mab)24B11 (closed squares), (ii) a control, non-catalytic monoclonalantibody (closed circles), and (iii) Mab 24B11 plus the immunizinganalog-ligand derivative (formula II, where R is methyl) as acompetitive inhibitor (closed triangles). The reactant ligand substratewas initially present at about 0.5 millimolar (mM) in a 50 mM phosphatebuffer (pH 7.0). The Mab 2411 or control monoclonal were present at 5micromolar (uM), and the inhibiting analog-ligand derivative was presentat 20 uM. The decrease in substrate concentration was determined by highperformance liquid chromatography (HPLC).

FIG. 3 contains two portions. The upper portion is a schematic depictionof the catalyzed lactone-formation reaction between a lactone synthaseand a reactant ligand using a usual Michaelis-Menten depiction. As isshown, the synthase and reactant ligand can reversibly form a complexanalogous to an enzyme-substrate complex. The equilibrium constant forthe formation is the Michaelis constant, K_(m). That complex can go onto form products and a regenerated synthase molecule via the rateconstant for the catalyzed reaction, k_(cat). The reactant ligand isalso shown as forming products by an uncatalyzed reaction with anuncatalyzed rate constant, k_(uncat). The lower portion of the figure isa graph showing three Lineweaver-Burk plots derivable from aMichaelis-Menten depiction for cyclization of the reactant ligand ofFIG. 1, where R is methyl, catalyzed by the monoclonal receptor lactonesynthase molecule secreted by hybriodoma 24B11. Velocities weredetermined spectrophotometrically by measuring the initial linearabsorbance change at 271 nanometers (nm).

The receptor [present at 2 micromolar (uM), as determined by Lowryassay, using a molecular weight of 150,000 daltons for an IgG molecule]was maintained in solution at 25 degrees C. prior to addition of thesubstrate reactant ligand. Reactions were initiated by addition ofvarying aliquots of a stock solution of reactant ligand substrate toprovide a substrate concentration of 20-100 uM. The substrate stocksolution was prepared by deprotection of the trimethylsilyl derivativeusing 5 percent citric acid in methanol, followed by dilution with 25 mMphosphate buffer, pH 7.0, to provide the desired concentration of stocksolution. The prepared stock solution as stored frozen at -80 degrees C.until immediately prior to use.

The first order rate constant (k_(uncat)) for the cyclization in theabsence of receptor molecules was measured similarly and used to correctthe initial rate data.

The data shown in the graphs are for the reaction with no inhibitorpresent (open circles), the reaction inhibited by 0.25 uM of theN-acetyl derivative of the analog-ligand of FIG. 1 (R is methyl; closedcircles), and the reaction inhibited by 0.50 uM of the same N-acetylderivative of the analog-ligand.

The ordinate is in units of reciprocal initial velocity, 1/V, in minutesper micromole (uM⁻¹ min). The abscissa is in units of reciprocalsubstrate concentration, 1/[S], in micromoles⁻¹ (uM⁻¹).

FIG. 4 is an exemplary graph illustrating the absorbance at 271 nm onthe ordinate versus time in minutes (min) for the cyclization of thereactant ligand of FIG. 3 catalyzed by the monoclonal receptor lactonesynthase molecule secreted by hybridoma 24B11.

The receptor was present at 20 uM in 25 mM phosphate buffer, pH 7.0, at25 degrees C. An aliquot of the ligand (present at 3.34 mM in phosphatebuffer, pH 7.0) calculated to provide a 40 uM solution of the ligand wasadded to the receptor solution at each of points A and B of the graph.

The average observed absorbance increase was shown to correspond toconsumption of 49±13 percent of the ligand after each injection,relative to a phenol standard.

FIG. 5 contains two graphs (A and B) that are portions of ¹ H nuclearmagnetic resonance (NMR) spectra of the lactone product of FIG. 1, whereR is methyl. The spectra were obtained in CDCl₃ at 360 MHz in thepresence of about one equivalent of the chiral lanthanide shift reagenttris[heptafluoropropylhydroxymethylene)-d-camphorato]europium (III).Chemical shifts are shown in parts per million (ppm) downfield fromtetramethylsilane (TMS).

Peak assignments (δ) and chemical shift differences between enantiomers(ΔΔδ) were determined as follows: Graph A δ9.45 and 9.68 (one of the CH₂NHCOCH₃, ΔΔδ=0.23); δ10.60 and 10.67 (NHCOCH₃, ΔΔδ=0.07); Graph B δ9.71and 9.94 (one of CH₂ NHCOCH₃, ΔΔδ=0.23; δ10.74 and 10.82 (NHCOCH₃,ΔΔδ=0.08).

For Graph A, the lactone was obtained by cyclization of thecorresponding reactant ligand substrate for 55 minutes at 25° C. in 25mM phosphate buffer, pH 7, in the presence of the lactone synthasereceptor molecule secreted by hybridoma 24B11, at an initial ratio ofreactant ligand substrate to receptor molecule of 9.2 mM:115 uM. ForGraph B, the lactone was obtained under the same reaction conditions,but without the presence of the lactone synthase receptor molecule. Whenpresent, the receptor molecule was removed by Centricon filtration,followed by methylene chloride extraction of the filtrate and columnchromatography on silica thereafter.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention relates to the production of antibodies andidiotype-containing polyamide (antibody combining site) portions inducedby an analog-ligand that mimics the stereoconfiguration of a transitionstate in the reaction sequence for the formation of a chemical bond suchas an ester or an amide bond, and particularly to the formation of alactone bond. The antibodies and idiotype-containing polyamides bind tothe amide- or ester-forming transition state of a preselected portion ofa ligand and exhibit catalytic properties.

Antibodies and enzymes are both proteins whose function depends on theirability to bind specific target molecules. Enzymatic reactions differfrom immunological reactions in that in an enzymatic reaction thebinding of the enzyme to its substrate typically leads to chemicalcatalysis, whereas a non-catalytic complex is the usual result ofantibody-antigen binding.

Enzymes are believed to catalyze the reactions of proteins or othersubstrates by combining with the protein (substrate) to stabilize thetransition state of the reaction. It is generally believed that the rateof an enzymatic reaction is increased relative to the rate of anon-enzymatic reaction because of the ability of the enzyme to stabilizethe transition state of the reaction; i.e., to reduce the free energy ofthe transition state, and thus, the free energy of activation, of thereaction [Jencks, W. P., Adv. Enzymology, 43, 219 (1975) and Pauling,L., Amer. Scientist, 36, 58 (1948)]. Support for this theory comes fromthe observation that substances that are thought to model the presumedtransition states are often strongly bound to the enzymes as competitiveinhibitors Leinhard, G., Science, 180, 149 (1973) and Wolfenden, R.,Acc. Chem. Res., 5, 10 (1972). It is further thought that the enzymeaccomplishes this lowering of the reaction free energy by binding thetransition state geometry of the reactant more strongly than it binds tothe corresponding substrate(s) or product(s).

This means that the intrinsic binding energy of the enzyme is muchgreater than can be measured from the binding of substrates or products.Essentially, the binding energy of the enzyme is utilized to perform thechemical reaction [Jencks, W. P , XVII International Solvay Conference(November 1983)].

The converse proposition is that a receptor that is prepared tooptimally bind a suitable analog of a transition state would function asa catalyst. The demonstration of this result as shown herein completesthe correlation of enzyme function and receptor structure, and providesa useful approach to devising artificial enzymes.

The basic idea behind bond formation catalyzed by a receptor asdescribed herein contemplates the use of analog-ligands in thepreparation of antibodies of predetermined specificity thatpreferentially bind to and thereby stabilize the transition state forbond formation upon binding to the specified reactant ligand. Theanalog-ligands simulate the stereochemical configuration of a highenergy transition state in bond formation to induce the production ofantibodies having the ability to bind to a reactant ligand when thatligand is in the appropriate stereochemical configuration, and tocatalyze bond formation in the bound reactant ligand. Following theterminology of enzymology, the reactant ligand can also be referred toas a ligand, substrate, a substrate ligand or a reactant ligandsubstrate.

Such preferential binding and stabilization results in a reduction inthe activation energy for the bond-forming reaction, thus meeting acriterion for catalysis. Antibodies that display this property can beobtained by immunization with synthetic analogs that are chemicallymodified to resemble the bonding characteristics of a substrate ligandundergoing bond formation; i.e., by immunization with transition stateanalogs of the particular bond-forming reaction.

The mechanism by which an antibody forms an ester or amide or other bondof a bound ligand can be thought of in terms of an "induced fit" model.As the loosely bound substrate distorts or rearranges to conform to thebinding geometry of the antibody combining site, stress can be relievedby chemical reorganization of the atoms involved in bond formation suchas an exemplary carbonyl carbon and an amine or alcohol of the ligandsuch that this reorganization leads to the formation of the desiredbond.

The transition state mimicked by the analog-ligand, and therefore boundby the antibody combining site, can stereochemically resemble thereactant, the product or a stereochemical configuration that is betweenreactant and products along the reaction coordinate. The "reactioncoordinate" is, like a transition state, a mental construct used bychemists to describe the progress of a reaction from a mechanisticviewpoint. The transition state is located at a potential energy peak asa reaction progresses. When graphed, potential energy is normally theordinate and the reaction coordinate is the abscissa.

The carboxylic acid ester and amide hydrolytic transition state analogsof Tramontano and Lerner and Pollack et al. discussed earlier resembleda transition state about midway between reactant and product. The workreported in WO 85/02414 did not appear to contemplate mimicking atransition state, but rather reactant, intermediate or product, and inthe only concrete example utilized the reactant for the principalimmunogen and a product-appearing portion of the reactant linkeddirectly to a carrier for a secondary immunogen.

The work described in detail hereinafter utilized a transition stateanalog-ligand as immunogen that resembled the desired product; i.e., thearrangement of the bonds of the analog-ligand that are involved in thebond-forming reaction resembled the arrangement of those bonds in theproduct more than they resembled the starting material or an arrangementabout midway between the reactant and product. Thus, the transitionstate analog-ligand utilized was in the form of a cyclic phosphonate sothat the induced receptor molecule would itself induce the acyclicreactant ligand into the cyclic configuration of the product and therebynot only lower the potential energy of activation by binding to thetetrahedral configuration of the carbonyl carbon during bond formation,but also reduce the rotational entropy of the reactant ligand upon thebinding of that ligand by the receptor.

Still further, the transition state mimicked the stereoconfiguration ofthe product so that only those reactant ligand molecules that could formthe product of the desired stereoconfiguration could be effectivelybound by the receptor synthase. As a consequence, substantially only oneof two possible stereoisomers was formed in the catalyzed reaction.

The term "receptor" is used herein to mean a biologically activemolecule that binds to a reactant ligand as well as to an analog-ligand.The receptor molecules of the present invention are antibodies,substantially intact antibodies or idiotype-containing polyamide(paratopic) portions of an antibody. Biological activity of a receptormolecule is evidenced by the binding of the receptor to its antigenicreactant ligand or analog-ligand upon their admixture in an aqueousmedium, at least at physiological pH values and ionic strengths.Preferably, the receptors also bind to the antigenic reactant ligand oranalog-ligand within a pH value range of about 5 to about 9, and ationic strengths such as that of distilled water to that of about onemolar sodium chloride.

Idiotype-containing polyamide portions (paratopes or antibody combiningsites) of antibodies are those portions of antibody molecules thatinclude the idiotype, and bind to the reactant ligand or analog-ligand.Such portions include the Fab, Fab' and F(ab')₂ fragments prepared fromantibodies by well-known enzymatic cleavage techniques. See for example,U.S. Pat. No. 4,342,566 to Theofilopoulos and Dixon, generally, andspecifically, Pollack et al., Science, 234, 1570 (1987), who reportedaccelerated hydrolytic rates for Fab fragments were the same as those ofthe native Ig. Inasmuch as the antibodies from which idiotype-containingpolyamides are obtained are described as raised against or induced byimmunogens, idiotype-containing polyamide receptors are also discussedas being "raised" or "induced" with the understanding that a cleavagestep is usually required to obtain an idiotype-containing polyamide froman antibody. Intact antibodies are preferred, however, and are utilizedherein as illustrative of the receptor molecules of this invention.

The receptors useful in the present invention are more preferablymonoclonal antibodies. A "monoclonal antibody" is a receptor produced byclones of a single cell called a hybridoma that secretes but one kind ofreceptor molecule that binds to a reactant ligand or analog-ligand. Thehybridoma cell is fused from an antibody-producing cell and a myelomacell or other self-perpetuating cell line.

Techniques for preparing the monoclonal antibodies of the presentinvention are well known. Such receptors were first described by Kohlerand Milstein, Nature, 256, 495 (1975), which is incorporated herein byreference. Monoclonal antibodies are typically obtained from hybridomatissue cultures or from ascites fluid obtained from mammals into whichthe hybridoma tissue was introduced. Both methods are described herein.

Monoclonal receptors are preferred herein because of their uniquespecificity in binding to a single epitope such as a particularstereoisomer of the immunizing analog-ligand and reactant ligand, aswell as their relatively higher specific catalytic activities ascompared to polyclonal antibodies. Polyclonal antibody preparations canalso be used herein, but typically have to be separated into fractionsthat bind to and catalyze a desired stereoisomer and those that bind tothe non-desired stereoiosomer.

Antibodies that bind to desired and non-desired stereoisomers can beseparated by affinity separation using an analog-ligand having thedesired or non-desired stereoiosomer as the affinity sorbant. Afteradmixture and maintenance of an antibody preparation with the affinitysorbant for a time sufficient for appropriate immunoreaction to takeplace, the affinity sorbant is separated from the remaining portion ofthe antibody preparation.

The separated, remaining antibody portion contains the antibodies thatbind to the desired stereoisomer where the non-desired stereoisomer isused as the affinity sorbant. On the other hand, where the desiredstereoisomer is used as the affinity sorbant, the separated, remainingantibody portion contains antibodies that bind to the non-desiredstereoisomer, whereas the antibodies that bind to the desiredstereoisomer are bound to the affinity sorbant. Those latter antibodiescan thereafter be isolated by usual techniques for separating boundentitites from affinity sorbants, such as washing the sorbant withglycinehydrochloride at pH 2.

In either event, mixed polyclonal antibodies that bind to a desiredconfiguration can be obtained. However, those antibody mixtures cancontain antibodies that do not catalyze the reaction. As a result, thespecific catalytic activity of such a mixed antibody preparation istypically lower than is the specific activity of a monoclonal receptor.

A "ligand" is defined herein as a molecule or complex that immunoreactswith or binds to a receptor molecule antibody combining site. Two typesof ligand are contemplated herein. A first is termed an analog-ligandand is used as an immunogen to induce preparation of receptor moleculesand as an inhibitor of the receptor molecule synthase-catalyzedreaction. The second is referred to as the ligand, reactant ligand,substrate, substrate ligand or reactant ligand substrate and is themolecule that undergoes the catalyzed reaction. The analog-ligand issubstantially inert to undergoing the catalyzed reaction.

As described herein, chemical analogs of ester ligands have beensynthesized that incorporate phosphonate moieties at a specific,predetermined site to mimic the atomic configuration of the transitionstate leading to the formation of an amide or ester bond. Such analogsare suitable candidates for this investigation because it is known thatphosphonamidates have been used as transition state analogs in theinhibition of proteolytic enzymes [Bartlett, et. al., Biochemistry, 22,4618 (1983)].

Short polypeptide chains can induce the production of antibodies thatrecognize and bind to a homologous protein at a predetermined specificsite. The present invention carries the earlier work with polypeptides amajor step forward. Here, the antibodies (receptors) are induced by animmunizing haptenic first molecule (the analog-ligand), and recognizeand bind not only to that first molecule, but also to a second, relatedmolecule (the reactant ligand). In binding that second molecule, thereceptor causes bond formation (which as demonstrated herein can becatalytic and stereospecific) of a preselected, ester or amide bond thatcorresponds in topology to the topology of the immunizing, haptenicfirst molecule. The correspondence in topology; i.e., size, shape(stereochemical configuration) and charge, provides a means forpreselecting the site at which bond formation of the reactant ligandoccurs.

Consequently, by synthesis of a relatively small, immunizing haptenicanalog-ligand, one can induce the production of receptor molecules thatrecognize, bind to and catalytically form a bond such as an ester oramide bond in a larger molecule that can contain a plurality of amide orester bonds. Thus, a receptor can be prepared that causes formation of aselected, predetermined bond in only one stereoisomeric form of aproduct molecule. The implication of this result is that one can conferthe activity of hitherto unknown synthases to immunoglobulins.

Furthermore, for syntheses of amides and esters, the activity of theantibody can be directed to any predetermined site at will bydesignating the amide or ester bond to be formed with thephosphonamidate or phosphonate configuration in the haptenicanalog-ligand used for immunization.

II. Transition State of Bond Formation Analog-Ligand and Reactant LigandDesign

Design of the analog-ligand flows backward from the structure of theproduct to be formed through the transition state for bond formation tobe mimicked, and then to the analog-ligand. Reactions that involve ringopening or ring closing provide illustrative examples of the generalconcept and are utilized herein as exemplary for a cyclic amide- orester-forming (lactam or lactone) reaction.

Amide or ester bond formation reactions catalyzed by an amide or estersynthase of the present invention are in some ways analogous totransacylation processes that are characterized by carbonyladdition-elimination mechanisms. The acyl group may, therefore, possessvarying degrees of tetrahedral character in this transition state. W. P.Jencks, Catalysis in Chemistry and Enzymology, ch. 10, (McGraw-Hill, NewYork, 1969). The enzymes that catalyze transacylation reactions might beexpected to bind well those analogs of the ligand having a tetrahedralconfiguration about the acyl center. This is true for serine proteases,where a covalent bond between the ligand and the enzyme is formedtemporarily [Westerik et al., J. Biol. Chem., 247, 8195 (1972); R. C.Thompson, Biochemistry, 12, 47 (1973) and Imperali et al., Biochemistry,25, 3760 (1986)], as well as for enzymes that catalyze the directhydration of amides or esters. The latter category is inhibited bycompounds with a tetrahedral configuration including a phosphate,phosphonate or phosphonamidate group in lieu of the scissile amide unit[Weaver et al., J. Mol. Biol., 114, 119 (1977) and Jacobsen et al., J.Am. Chem. Soc., 103, 654 (1981)].

Naturally occurring and synthetic substances containing phosphorus havebeen studied as inhibitors of metallopeptidases. In these enzymes, thetransition state would appear to contain the hydrated amide in thecoordination sphere of the metal ion [W. N. Lipscomb, Acc. Chem. Res.,15, 232 (1982)]. A complete picture of a transition state analog mightthen have the phosphono group of an inhibitor as a ligand to a metal ionor some other polarizing site [Weaver et al., J. Mol. Biol., 114, 119(1977) and Christianson et al., J. Am. Chem. Soc., 108, 545 (1986)]. Therole of the metal ions in metallopeptidases, however, is not clearlyunderstood. The metal may have a multiple function in amide hydrolysiswhere proton transfer steps among the tetrahedral intermediates may berate-limiting [L. M. Sayre, J. Am. Chem. Soc., 108, 1632 (1986)].

The formation of a carboxylic amide or ester is a simple example ofacylation that can also be approximated by the phosphonate-containinganalog of the tetrahedral transition state. The binding of thephosphonate group may describe a stabilizing interaction in thetransition state which would lead to catalysis. Amide and esterformation reactions generally do not proceed at convenient spontaneousrates under ambient conditions that are suitable for antibodies.Therefore, bond formation (or loss of substrate) can be readilydetected.

The structures of the analog-ligands and ligands for this investigationwere selected according to certain criteria. These included theavailability and stability of the organophosphorus precursors, thecorresponding carboxylic acid substrate, the convenience of the chemicalsynthesis for its preparation, and the adaptability to diverse schemesfor immunological presentation.

An exemplary basic molecular unit for the analog-ligand that providesthe structural features necessary for inducing production of a lactonesynthase is the phosphorus-containing cyclic compound of formula IIIthat is shown below: ##STR3##

wherein X is O or NH;

R is selected from the group consisting of hydrogen, C₁ -C₆ lower alkyland a linking group alone or bonded to an antigenic carrier; and

R¹ is selected from the group consisting of hydrogen C₁ -C₆ lower alkyl,benzyl and phenyl.

As is apparent from the above formula, when X is an oxygen atom (O), theanalog-ligand is a lactone (cyclic phosphonate), whereas when X is anitrogen atom bonded to a hydrogen atom (NH) the analog-ligand is alactam (cyclic phosphonamidate).

An analog-ligand of the above formula can be linked to an antigeniccarrier, molecule through an appropriate linking group R. Specificlinking groups and antigenic carrier molecules are discussedhereinafter.

A particularly preferred analog-ligand has a structure represented byformula II, below: ##STR4##

wherein R is selected from the group consisting of hydrogen, C₁ -C₆lower alkyl, and a linking group alone or bonded to an antigenic carriermolecule.

When used as an immunogen, the R group of the haptenic analog-ligand ispreferably a linking group bonded to an antigenic carrier, as describedhereinafter. When used as an inhibitor, as in the studies shown in FIG.2, the R group is preferably methyl (C₁ alkyl).

Examination of the structures of the analog-ligands of formulas III andII reveals that there are two centers of chirality, at the phosphorusatom and at the carbon atom adjacent to the X and O of the formulas,respectively; i.e., the alpha-carbon of the alcohol or amine portions ofthe ester or amide. Thus, the analog-ligands depicted can exist as atotal of four stereoisomers (diastereomers); i.e., two pairs ofenantiomers. Further pairs of enantiomers are possible where R¹ and Rcontain chiral centers. Data from nuclear magnetic resonancespectroscopy indicated that the ring closure reaction that formed theprimary amine corresponding to formula II was stereospecific as to thephosphorus atom, and only one diastereomer (a single pair ofenantiomers) was in fact formed.

The reactant ligand (substrate) structurally capable of forming an amideor ester bond and that contains a carbonyl group carbon atom and anamine or alcohol group can be a single molecule that contains both ofthe reactive functionalities, or those functionalities (carbonyl carbonand amine or alcohol) can be on separate molecules. The singular form ofthe word "ligand" is utilized for both the single and two moleculespecies since once bound, either type of those entities behaves as asingle molecule.

A preferred reactant ligand capable of forming a lactam or a lactonecorresponding to the analog-ligand of formula III has a structure offormula IV, below: ##STR5##

wherein X and R¹ are as before-described, and R² is hydrogen for C₁ -C₆lower alkyl, preferably methyl (C₁).

A particularly preferred ligand of formula IV is the ligand that has thestructure of formula I, below: ##STR6##

wherein R² is as described before.

It is noted that the reactant ligands of formulas IV and I also containa chiral carbon atom. Again, that chiral atom is the carbon bondeddirectly to the amino or hydroxyl group of the amine or alcohol portionof the amide or ester to be formed; i.e., the alpha-carbon. In view ofthe chirality of that atom, the reactant ligand substrates depicted informulas IV and I normally exist as a racemic modification of twoenantiomers. Further enantiomers (diastereomers) can also exist forthose formulas, depending on the structures of R¹ and R² as notedpreviously.

The structures depicted in formulas IV and I depict the four differentgroups bonded to the chiral atom, whereas the structures of formulas IIIand II omit the hydrogen atom (H) bonded to that atom for improvedclarity in viewing the formulas. All four of those formulas omit thehydrogen atoms between the phosphorus atom or carbonyl carbon atom andthe chiral atom for the same reason. The individual carbon atoms bondedto those omitted hydrogens and the chiral carbon itself are shown as thevertices of the lines joining the phosphorus atom or carbonyl carbonfollowing procedures for depicting such atoms as is customary in organicchemistry.

An antibody molecule and its combining site are chiral, being a polymerof naturally occurring optically active L-amino acid residues. Becauseof the particular make-up of amino acid residues of the binding site andthe folding of those residues, an antibody combining site can alsopreferably bind to one of two stereoisomers. This fact is shown hereinby the preferential binding and reaction with one of a pair ofenantiomers as compared to the other. The present invention makes use ofthe exquisite binding properties of antibody combining sites to catalyzethe synthesis of one stereoiosmeric product, here an enantiomer, ascompared to the other stereoisomeric product that might otherwise alsobe formed by the catalytic reaction.

Thus, one aspect of the present invention contemplates a method forcarrying out a stereoselective synthesis to prepare a preselectedproduct containing relatively more of one stereoisomer than anotherstereoisomer that might be formed. In carrying out the synthesis, areactant ligand structurally capable of forming the product and aneffective amount of a synthase molecule are admixed in an aqueous mediumto form a reaction mixture.

The reaction mixture so formed is maintained for a time periodsufficient for the stereoisomeric product to form, e.g. minutes to days.The desired stereoisomeric product is thereafter typically recoveredfrom the remainder of the reaction mixture.

That remaining reaction admixture can also include the synthase,unreacted reactant ligand of the desired stereoconfiguration (where thereaction has not gone to completion), unreacted reactant ligand of thenon-desired stereoconfiguration (where a mixture such as racemicmodification of two enantiomeric reactant ligands is used), and productof the non-desired stereoconfiguration (where an uncatalyzed reactiontakes place or where complete stereospecific catalysis is not obtained).The desired product need not be recovered in instances where it is to beused as present in the reaction mixture, such as where a subsequentreaction is to be performed.

The reactant ligand utilized is capable of forming at least twostereoisomers of the product; i.e., cis/trans isomers or enantiomers. Itis to be understood that more than two stereoisomers such asdiastereomers can be formed in several reactions contemplated herein.When more than one pair of stereoisomers can be formed, at least one ofthe stereoisomers is formed in preference to the others. Wherediastereomers are involved, the at least one preferentially formedstereoiosomer can include a pair of enantiomers.

The synthase molecule comprises a receptor molecule, preferablymonoclonal, that contains an antibody combining site capable ofstereospecifically catalyzing the formation of the product. Thatcombining site binds to: (a) substantially only one stereoisomer of thereactant ligand and (b) a ligand structurally analogous to a transitionstate that leads to one stereoisomer of the product; i.e., theanalog-ligand.

Useful synthase molecules are prepared by immunizing an animal such as amouse with an analog-ligand as immunogen to induce production ofantibodies to the analog-ligand. The presence of antibodies that bind tothe analog-ligand can be determined by a simple screening procedure suchas the enzyme-linked immunosorbant assay (ELISA) described herein. Oncea positive binding result is obtained for a polyclonal antibodypreparation, an antibody preparation that binds preferentially to thedesired stereoisomer can be obtained by a method described before.

Where a monoclonal synthase is desired, a polyclonal antibodypreparation can be screened for general binding reactivity with theanalog-ligand, and hybridomas thereafter prepared by standard techniquesfrom a myeloma line and appropriate cells of the animal used to preparethe positive-binding antibody preparation. Since the monoclonalantibodies secreted by the prepared hybridomas should also be screenedfor immunoreaction with the analog-ligand, the first screening ofpolyclonal antibodies is not always necessary.

Thus, the monoclonal antibodies secreted by the produced hybridoma aretypically screened first for those that bind to a desired stereoisomerof the analog-ligand, and thereafter the antibodies that bind arescreened for the capacity to catalyze the desired reaction of thereactant ligand. Since the desired result is catalysis of a particularstereoselective reaction, the screening for binding to an analog-ligandof the desired stereochemistry can also be omitted, and the hybridomasecretions screened only for the ability to stereospecifically catalyzethe reaction.

The hybridoma(s) that secrete a useful monoclonal antibody is (are)thereafter grown further to produce more of a useful antibody.

In preferred practice, the preselected stereoisomeric product is a pairof enantiomers. In a particularly preferred practice, the synthasemolecule is a carboyxlic acid amide or ester synthase.

A method of forming a preselected enantiomeric carboxylic acid amide orester product that contains an excess of one enantiomer over the otherenantiomer is also contemplated herein. Here, an enantiomeric reactantligand pair structurally capable of forming the enantiomeric product;i.e., a reactant ligand racemic modification, is admixed with aneffective amount of a carboxylic acid amide or ester synthase in anaqueous medium to form a reaction mixture.

The reactant ligand contains a carbonyl group carbon atom and an amineor alcohol group structurally capable of forming the preselectedcarboxylic acid amide or ester product. As noted earlier, the reactivegroups, here a carbonyl group and an amine or alcohol group can be inthe same molecule as described in detail herein, or in separatemolecules as where the carbonyl group carbon atom is in a compound suchas that of formula II where R is methyl and the amino nitrogen is thatof aniline, for example, that is admixed to form an anilide by acatalyzed reaction. Aside from a doubly bonded oxygen atom, the carbonylcarbon atom is typically singly bonded to an alpha-carbon atom andsingly bonded to another, leaving, group atom such as the oxygen of thealcoholic portion of an ester, the nitrogen of the amine portion of anamide, a halide, the nitrogen of an azide group or the sulfur of athiolester. The reactant ligand can thus be referred to as containing acarbonyl group that is activated toward amide or ester formation ascompared to the carbonyl group of a carboxylic acid.

The amide or ester synthase molecule comprises a receptor molecule,preferably monoclonal, that contains an antibody combining site capableof catalyzing the formation of the preselected enantiomeric amide orester product. That combining site binds to: (a) substantially only oneenantiomer of the enantiomeric ligand pair, and (b) a ligandstructurally analogous to one enantiomer of a transition state leadingto the preselected amide or ester product; i.e., the analog-ligand.

The enantiomer of the analog-ligand that is bound by the combining sitecontains a tetrahedrally bonded phosphorus atom that is located at theposition occupied by the carbonyl group carbon atom of the carboxylicacid amide or ester product. That tetrahedrally bonded phosphorus atomis itself bonded directly to: (i) the alpha-carbon of the acid portionof the analog-ligand by a single bond; (ii) a first oxygen atom that isdoubly bonded to the phosphorus atom; (iii) a second oxygen atom that issingly bonded to the phosphorus atom, and is singly bonded to a radicalselected from the group consisting of hydrogen, C₁ -C₆ lower alkyl,benzyl and phenyl; and (iv) a third oxygen atom or a nitrogen atom thatis singly bonded to the phosphorus atom, and is also singly bonded tothe alpha-carbon of the amine or alcohol portion of the analog-ligand.It is to be understood that the nitrogen atom can be of a primary amineor an appropriately substituted secondary amine. This is true for all ofthe amine portions discussed herein, although the preferred exemplaryanalog-ligand and reactant ligands of formulas III and IV, respectively,are shown as primary amines. Tertiary amines do not form amides.

For formation of a cyclic amide or ester as is prepared illustrativelyherein, there are not distinct acid and amine or alcohol portions of themolecule. However, those skilled in organic chemistry will understandthat amides and esters must by definition contain acid and amine oralcohol portions. Thus, an imaginary line of demarcation can be drawnfor such molecules that includes at least the carbonyl carbon and itsdirectly bonded alpha-carbon in the acid portion of the molecule andincludes the amino or hydroxyl group and its directly bondedalpha-carbon in the amine or hydroxyl portion of the molecule.

The reaction mixture so formed is maintained for a time periodsufficient for the preselected carboxylic acid amide or ester productenantiomer to form, as already noted. That product is again preferablyrecovered, although such recovery is not required.

Preferably, the amide or ester synthase is a lactam or lactone synthase.This synthase is also preferably an intact antibody. Most preferably,the ester or amide synthase is secreted by hybridoma 24B11 that wasdeposited with the American Type Culture Collection (ATCC) of Rockville,Md., pursuant to the Budapest Treaty on Aug. 4, 1987 and bears theaccession number HB9488.

A method of forming a preselected carboxylic acid amide or ester productis also contemplated. In accordance with this method, a reactant ligandstructurally capable of forming the amide or ester product is admixedwith an effective amount of an amide or ester synthase in an aqueousmedium to form a reaction mixture.

The reactant ligand contains a carbonyl group carbon atom and an amineor alcohol group structurally capable of forming the preselectedcarboxylic acid amide or ester. The carbonyl carbon atom is bonded to anoxygen atom, an alpha-carbon atom and an atom of a leaving group aspreviously described for the reactant ligand for synthesis of anenantiomeric ester or amide.

The amide or ester synthase molecule comprises a receptor molecule,preferably monoclonal, that contains an antibody combining site capableof catalyzing the formation of the preselected amide or ester product.That combining site binds to the reactant ligand and also to a ligandstructurally analogous to the preselected amide or ester product; theanalog-ligand.

The analog-ligand contains a tetrahedrally bonded phosphorus atomlocated at the position of the carbon atom of the carbonyl group of thepreselected carboxylic acid amide or ester product. That tetrahedrallybonded phosphorus atom is bonded directly to: (i) the alpha-carbon ofthe acid portion of the analog-ligand by a single bond; (ii) a firstoxygen atom that is doubly bonded to the phosphorus by a single bond;(iii) a second oxygen atom that is singly bonded to the phosphorus atomand is singly bonded to a radical selected from the group consisting ofhydrogen, C₁ -C₆ lower alkyl, benzyl and phenyl; and (iv) a third oxygenatom or a nitrogen atom that is singly bonded to said phosphorus atom,and is also singly bonded to the alpha-carbon of the alcohol or amineportion of the analog-ligand.

The reaction mixture is maintained for a time period sufficient to formthe preselected amide or ester product, as already noted. That productcan thereafter be recovered, as discussed previously for other products.

The synthase molecule is preferably a lactam or lactone synthase. Thesynthase can also catalyze the amide or ester bond formation chirally toform relatively more of one of a pair of stereoisomeric products such asenantiomers, than the other of the pair. Thus, an amide or estersynthase useful in a before-described method can also be utilized inthis method.

A synthase molecule useful in the method described immediately above orin any of the methods previously described can be an intact antibody,which is preferred, or a smaller, antibody combining site-(paratope-)containing portion of an antibody such as Fab or Fab' portion of anantibody.

Each of the previously described synthetic methods utilizes an aqueousmedium as a portion of the reaction admixture. That medium typicallycontains water and buffer salts. In addition, the medium can containother salts such as sodium choride, as well as water-soluble calcium andmagnesium salts as are frequently found in protein-containing media.Other water-soluble polyvalent metal salts such as iron and cobalt saltscan also be present and are useful complexing agents where the reactantligand is comprised of two separate molecules. Organic solvents such asmethanol, ethanol, acetonitrile, dimethyl sulfoxide, dioxane,hexamethylphosphoramide and N,N-dimethylforamide can also be present.Surface active agents that emulsify the reactant ligand and synthasemolecule can also be present. The critical feature of ingredientspresent in the aqueous medium is that those ingredients notsubstantially interfere with or inhibit the catalytic reaction as bydenaturation of the synthase molecule. Additionally, the aqueous mediumis substantially free from salt, proteins generally, and enzymes,specifically, that inhibit the bond-forming reaction catalyzed by thesynthase molecule.

The aqueous medium typically has a pH value of about 5 to about 9, andpreferably about pH 6.0 to about 8.0. pH values greater and less thanthose recited values can also be utilized so long as the catalyzedreaction is again not substantially interfered with or inhibited.

The catalytic reactions are typically carried out at ambient roomtemperature; i.e., at about 20 to about 25 degrees C., and at an ambientatmospheric pressure. However, temperatures down to about the freezingpoint of the aqueous medium and up to about the boiling point of themedium at atmospheric pressure can also be used. As is known, proteinssuch as the synthase molecule tend to denature at elevated temperaturessuch as those at which an aqueous medium boils, e.g. at about 100degrees C., and thus temperatures below about 40 degrees C. arepreferred. As is also well known, reactions that follow multimolecularkinetic expressions decrease in rate as the temperature decreases. Thus,a minimal temperature of about 15 degrees is preferred.

The reactant ligand is present in a reaction mixture in an amount up toits solubility in the aqueous medium. A two phase system that includesinsoluble reactant ligand can also be used, but normally is not so used.Normally used concentrations of the reactant ligand are about 0.1micromolar (uM) to about 10 millimolar (mM), with that amount also beinga function of the solubility of the reactant ligand in the solventmedium. Where the product is desired, per se, relatively higherconcentrations are used as compared to lower concentrations where areaction mechanism or reaction kinetics are to be studied.

An effective amount of the synthase molecule is also present. Thateffective amount is typically a catalytic amount; i.e., the synthase isused at a molar ratio to the reactant ligand of about 1:5 to about1:10,000. The ratio of synthase molecule to reactant ligand typicallydepends upon the specific activity of the synthase molecule toward thesubstrate ligand and the purpose of the user in running the reaction.Thus, where the product is desired, a relatively higher concentration ofsynthase and higher synthase to reactant ligand ratio are used. Wherethe reaction mechanism or kinetics of the reaction are being studied, alower concentration and ratio are typically used. A stoichiometricamount of synthase or less can also be used, but since the synthase is acatalytic molecule, use of even a stoichiometric amount can be wasteful.Thus, at least a catalytic amount of the synthase is utilized.

The present invention also contemplates a receptor molecule thatexhibits synthase activity and comprises a receptor moecule that ispreferably a monoclonal receptor.

In one embodiment, the receptor has an antibody combining site capableof catalyzing stereoselective synthesis and can catalyze the preparationof a preselected product that contains relatively more of onestereoisomer than the other stereoisomer. The antibody binding sitebinds to: (a) substantially only one stereoisomer of a reactant ligandthat is structurally capable of forming the product and also (b)substantially only one steroisomer of an analog-ligand that isstructurally analogous to a transition state leading to one stereoisomerof the product.

As noted earlier, steroisomers can be geometric isomers or opticalisomers. In the reactions discussed in detail hereinafter, the reactantligand used was an enantiomeric pair that reacted to form substantiallyonly one of the possible enantiomeric products in the catalyzedreaction. The analog-ligand used to induce production of the monoclonalsynthase receptor molecule was itself one of a pair of diastereomers;i.e., an enantiomeric (optical isomer) pair of geometric isomers inwhich the phenoxy group of the phosphonate group could be in a 1,3-cisor -trans relation to the aminomethyl group.

A useful synthase molecule can thus selectively catalyze a reaction thatpreferentially leads to formation of one cis or trans isomer over theother. Similarly, a useful synthase molecule can selectively catalyze areaction that preferentially leads to the formation of one enantiomerover the other enantiomer.

As already noted, a synthase molecule that contains an antibodycombining site capable of catalyzing the formation of a preselectedenantiomer of a carboyxlic acid amide or ester product is alsocontemplated herein. That antibody combining site binds to substantiallyonly one enantiomer of an enantiomeric reactant ligand pair, and to aligand structurally analogous to one enantiomer of a transition stateleading the preselected amide or ester product. The enantiomer of theanalog-ligand that is bound by the combining site has a tetrahedrallybonded phosphorus atom located at the position occupied by the carbonatom of the carbonyl group of the carboxylic acid amide or esterproduct. The other atoms bonded to that phosphorus and the number ofbonds between each of those atoms and the phosphorus atom are aspreviously described.

It is to be noted that the stereoconfiguration of the reactant moleculeneed not be that of the product that is formed preferentially, whetherthat product is an optical or geometric isomer. The stereoconfigurationof the reactant ligand and that of the analog-ligand also need not bethe same. Rather, the steroconfigurational relationship between thereactant ligand, analog-ligand and product is a function of theparticular reaction that is being catalyzed.

More specifically, where an atom involved in the bond-forming reactionitself has a particular stereoconfiguration and the catalyzed reactioninvolves a displacement such as a nucleophilic displacement thatproceeds by an S_(N) 2 mechanism, the stereoconfiguration at that atominverts to the opposite configuration. When such an atom is also achiral center, the reactant ligand possesses a first stereoconfigurationwhereas an analog-ligand that resembles a product-like transition statehas a second, inverted stereoconfiguration of the product.

On the other hand, where the atom at which bond formation takes placedoes not possess two stereoconfigurations or where an inversion ofconfiguration is not involved in the bond-forming reaction, thestereoconfiguration of the reactant ligand, analog-ligand and productare substantially the same.

This is the case for the lactone formation described in detail hereinsince the carbonyl carbon of the ester reactant ligand of formula I,while involved in the bond-forming reaction, does not exist in twostereoconfigurations, whereas the alpha-carbon of the alcohol portion ofthat ligand has d and l isomeric forms, but no inversion or otherstereochemical change is involved at that carbon. The phosphonate groupof the analog-ligand of formula II can exist in two stereochemicalforms, as can the alpha-carbon of the alcohol portion of the cyclicphosphonate. However, as noted herein, only one of the phosphonatediastereomers formed when that analog-ligand was prepared. Thus, theremaining center of stereoisomerism, the alpha-carbon of the alcoholportion of the cyclic phosphonate, had the same stereoisomeric forms asthe analogous alpha-carbon of the reactant ligand alcohol portion.

The present invention still further contemplates a molecule exhibitingamide or ester synthase activity that comprises a receptor molecule. Thepreferably monoclonal receptor contains an antibody combining sitecapable of catalyzing the formation of a preselected carboxylic amide orester bond. The combining site binds to: (a) a ligand containing acarbonyl carbon atom and an amine or alcohol group that are structurallycapable of forming the preselected carboxylic amide or ester bond; and(b) a ligand structurally analogous to the preselected amide or ester;the analog-ligand having a tetrahedrally bonded phosphorus atom locatedat the position occupied by the above-mentioned carbonyl carbon atom ofthe preselected carboxylic amide or ester bond of the before-mentionedligand. The tetrahedrally bonded phosphorus atom is itself directlybonded to: (i) the alpha-carbon atom of the acid portion of the ligandby a single bond; (ii) a first oxygen atom that is doubly bonded to thephosphorus atom; (iii) a second oxygen atom that is bonded to thephosphorus atom by a single bond, and is singly bonded to a radicalselected from the group consisting of hydrogen, C₁ -C₆ lower alkyl,benzyl and phenyl; and (iv) a third oxygen atom or a nitrogen atomsingly bonded to the phosphorus atom, and is also singly bonded to thealpha-carbon atom of the amine or alcohol portion of the ligand.

In preferred practice, the molecule exhibits lactone synthase activity.Thus, the ligand containing the carbonyl carbon is capable of forming alactone, and the analog-ligand is a cyclic phosphonate, as exemplifiedby 2-phenoxy-2-oxo-6-(acetamidomethyl)-1,2-oxaphosphorinane.

The phrase "structurally capable of forming a preselected carboxylicamide or ester bond" and similar phrases using the words "structuallycapable" are utilized herein to indicate that the structure of theligand is such that it will permit and not inhibit, as by sterichinderance or by the absence of necessary reactive groups, the bond tobe formed.

III. Coupling of Compounds to Protein Carriers

Conjugates of haptenic analog-ligand molecules with protein carrierssuch as keyhole limpet hemocyanin (KLH) can be prepared, for example, byactivation of the carrier with a linking agent such as MBS(m-maleimidobenzoyl-N-hydroxy succinimide ester), and coupling to thethiol group of the analog-ligand. See, for example, Liu et al.,Biochem., 80, 690 (1979). As is also well known in the art, it is oftenbeneficial to bind a hapten to its carrier by means of a linking groupthat is reacted first with the hapten and then the resultinghapten/linker is reacted with the antigenic carrier. Thus, the hapten isactivated rather than the carrier. The acyl chloride portion of theselinkers typically reacts first.

In addition to MBS, glutaraldehyde and other well known linking groups,two other linking groups have been found useful. These linkers areN-hydroxysuccinimidyl glutaryl chloride and N-hydroxysuccinimidyladipoyl chloride whose syntheses are described herein.

Useful antigenic carriers are well known in the art and are generallyproteins themselves. Exemplary of such carriers are keyhole limpethemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine serumalbumin or human serum albumin (BSA or HSA, respectively), red bloodcells such as sheep erythrocytes (SRBC), tetanus toxoid, cholera toxoidas well as polyamino acids such as poly(D-lysine:D-glutamic acid), andthe like.

The choice of carrier is more dependent upon the ultimate intended useof the antigen or immunogen than upon the determinant portion of theantigen, and is based upon criteria not particularly involved in thepresent invention. For example, a carrier that does not generate anuntoward reaction in the particular laboratory utilized animal should beselected.

The exemplary immunogenic conjugate was prepared from the cyclicphosphonate by adapting its synthesis to incorporate a straight chain ofcarbon atoms on the methylene amino group of the lactone as a spacingelement. It was concluded that the flexible carbon chain of an glutarateappendage would reduce any bias to immunoreactivity due to theconformational constraint imposed by covalent attachment to the carrierprotein. The bifunctional reagent prepared for this purpose alsodelivers the preactivated carboxyl group for linkage via amide bondformation with the lysine residues of the carrier. The particularcoupling method used in this study is further described herein. Thecyclic phosphonate was coupled to keyhole limpet hemocyanin (KLH)through a methylene amino group.

According to the present invention, the intermediate linking agent ispreferably succinimidyl glutaryl chloride that was prepared as describedhereinafter.

IV. Separation of Stereoisomers

The present invention also provides a method of separating stereoisomersfrom each other. Here, a mixture of stereiosomers and a receptormolecule containing an antibody combining site that immunoreacts withsubstantially only one of those stereoisomers are admixed in an aqueousmedium to form an admixture. The admixture is maintained for a timeperiod sufficient for the receptor to bind to one of the steroisomersand to form an immunoreactant of the receptor and one stereoisomerwithin the admixure, and a remaining admixture. The immunoreactant isthereafter separated from the remaining admixture. In preferredpractice, the bound stereoisomer is separated from the receptor of theimmunoreactant.

In keeping with the preceding discussion, the stereoisomers can begeometric, cis/trans, isomers or optical, enantiomeric isomers.Similarly, the aqueous medium utilized for separation can be an aqueousmedium as is used for a carrying out a before-discussed reaction. Theconcentrations and ratio of receptor and stereoisomers can be asdescribed previously, although a stoicheometric ratio and relativelydilute concentrations are preferred.

Where separations such as those discussed above are carried out, it ispreferred to link the receptor molecule to a water-insoluble solid phasematrix to form a solid phase sorbant. Such solid phase sorbants areknown in the art as affinity sorbants, and their methods of preparationare well known.

Every material has at least some water-solubility. As a consequence, theterm "water-insoluble" is used herein in its usual sense to mean thatthe matrix and sorbant made therefrom are recovered substantially intactand in substantially the same amount as admixed with the aqueous mediumwhen the separation is carried out. The matrix and sorbant are typicallyswellable in water, and can form a gel-like solid phase and still bewithin the purview of a water-insoluble material as contemplated herein.

A particularly preferred affinity sorbant contains a before-describedreceptor linked to a particulate water-insoluble matrix comprised ofcross-linked agarose. Particularly preferred is a cross-linked agarosesuch as cyanogen bromide-activated Sepharose 4B-CL (Pharmacia FineChemicals, Piscataway, N.J.) which is readily linked to amine-containingmaterials such as receptors to form the solid phase sorbant.

Sepharose 4B-CL is utilized herein as an exemplary solid phase matrix.However, additional particulate and monolithic solid phase matrices arealso useful herein. Exemplary of such matrices are Sepharose 6B and 4B,glass beads, and the inner and outer surfaces of hollow fibers as areuseful in hemodialysis or ultrafiltration. In addition to matricesspecifically mentioned herein, several suitable particulate (beaded)matrices are listed in the 1984 Sigma Chemical Company catalogue atpages 98 to 113. Typically, any water-insoluble solid phase matrix thatreacts with an amino group or a carboxy group is suitable.

Methods of affixing a receptor molecule to the matrix are also wellknown by skilled artisans and need not be dealt with in detail herein.Illustratively, however, such methods include use of activated carboxylgroups as are provided by cyanogen bromide treatment ofglucose-containing solids and chemical reactions using water-solublecarbodiimide technology, glutaraldehyde linking and the like.

In addition, U.S. Pat. No, 4,357,311 to Schutt discloses a method forpreparing an activated microporous substrate to which an antibody can becovalently bonded through trichloro-triazine to yield an activatedsubstrate. That method can also be used herein. Further, numerousmethods for immobilizing enzymes that are applicable for affixing areceptor to a matrix are discussed in Enzyme Technology, published byNoyes Data Corporation (1983) at pages 38 to 59.

Where the desired stereoisomer is the material bound by the receptor,the immunoreactant formed of the receptor and stereoisomer is firstseparated from the remaining admixture. Thereafter the boundstereoisomer is separated from the receptor using well known techniques,and typically recovered. Where the desired stereoisomer is not theisomer bound by the receptor, separation of the immunoreactant from theremainder of the admixture provides an aqueous composition containing anenriched amount, and preferably only, the desired stereoisomer.

Precipitation and centrifugation techniques well known inimmunochemistry are also useful for separating the immunoreactant fromthe remaining admixture. These techniques are useful with a solid phasesorbant and also where the admixture is substantially homogeneous.

A wide variety of steroisometric materials can be separated by a methodof this invention. So long as the steroisomers can be dissolved in theaqueous medium and the receptor molecule can immunoreact with whicheverisomer it preferentially reacts with under the conditions in which thesteroisomers are dissolved in the aqueous medium, the isomers can beseparated. Successive separations in which the stereoisomers areseparated and the separation steps are repeated can be utilized toimprove the stereoisomeric purity of separated isomers.

Exemplary stereiosomers that can be separated include relatively smallmolecules such as the analog-ligand enantiomers and lactone enantiomersdiscussed herein. Synthetic natural product analogs, agonists andantagonists such as prostaglandin derivatives, terpenes, drugs such asthalidomide, gluethimide, nalorphine, digoxin and other steroidalcompounds can be separated by this method. Still further, a polypeptidethat contains a D-amino acid residue can be separated from a polypeptidecontaining the same sequence having an L-amino acid replacement for thatD-amino acid residue.

The receptor molecules utilized in this method are preferablymonoclonal, for the reasons discussed previously. A synthase molecule asherein described can also be used where appropriate; i.e., where thestereoisomers of the analog-ligand or product are desired, but generallynot where stereisomers of the reactant ligand are to be isolated.

Thus, the receptor utilized for this method need not, and preferablydoes not, catalyze a reaction of either stereoisomer. Rather, it needonly preferentially bind to one stereoisomer over the other. Suchreceptor molecules are relatively easy to prepare.

For example, where the desired isomer to be separated is a syntheticform of a naturally occurring compound such as progesterone, thenaturally occurring compound, which exists in nature as one of severalpossible stereoisomers, is used as the immunogen, and is typicallylinked to an antigenic carrier molecule such as keyhole limpethemocyanin (KLH), as by reaction through the progesterone 17-keto group.After immunization to induce production of anti-progesterone antibodies,polyclonal antibodies that immunoreact with the immunogen can beseparated from the remaining antibody preparation by usual affinitychromatographic techniques.

After separation of the bound anti-progesterone antibodies from theimmunosorbant, those antibodies can be reacted with a second affinitysorbant, and the bound and unbound antibodies separated. The separatedantibodies that bound to the first sorbant but not to the second aretherefore specific for the stereoconfiguation of the immunogen, whereasthose that bound to the second affinity sorbant bind to bothstereoisomers. The antibodies specific for the stereoconfiguration ofthe immunogen can thereafter be used to separate the stereoisomers asdescribed before.

A somewhat less specific antibody preparation can also be prepared byscreening only against the molecule of the stereoconfiguration otherthan that of the immunogen as with an affinity column. Here, the unboundantibodies immunoreact with the molecule of the desiredstereoconfiguration or with an extraneous molecule such as the carrier.That antibody preparation can, nevertheless, be used to separate thestereoisomers.

In another procedure, a mixture of the stereoisomers is used as theimmunogen, and the resulting antibodies are screened and separated basedon their ability to bind to one or both isomers.

Monoclonal receptors useful in this method are readily prepared asdescribed before, using a molecule of the desired stereoconfiguration,its stereoisomer or a mixture of stereoisomers as immunogen, generallyas a conjugate to an antigenic carrier. Once secreting hybridomas areformed, one or more useful hybridomas and their monoclonal receptors canbe obtained by separately screening the secreted monoclonal antibodiesagainst each of the stereoisomers as in an ELISA, and selecting for usea hybrioma that secretes receptor molecules that immunoreact withsubstantially only one of the stereoisomers and not with the otherstereoisomer. The useful hybridoma is thereafter typically cloned,injected into the peritoneal cavity of an appropriate animal such as amouse, where the hybridoma was from compatible mouse cells, and theuseful monoclonal receptor, as an intact antibody, recovered from theascites fluid produced.

The present invention is further illustrated by the examples thatfollow, which are not intended to be limiting.

EXAMPLE 1 Preparation of Succinimidyl Adipoyl Chloride (Linking Agent)

A solution of adipic acid monomethyl ester (5.4 g, 33.3 mmol) in thionylchloride (15 ml) was heated at 40 degrees C. for 2 hours. The mixturewas then concentrated and distilled in vacuo (boiling point 119 degreesC. at 20 mm Hg) to provide 3.58 g (60 percent yield by weight) of theacid chloride methyl ester. This was dissolved in 20 ml ofdichloromethane and N-hydroxysuccinimide (2.75 g, 24.0 mmol) was added,followed by triethylamine (4.2 ml, 30 mmol). The mixture stirred for 10minutes then diluted with ethyl acetate and washed with 0.5M HCl andbrine. The solution was dried over anhydrous magnesium sulfate, filteredand concentrated to give 4.5 g (87.5 percent yield by weight) of methylsuccinimidyl adipate as a colorless oil.

Proton NMR (in CDCl₃): delta 3.73 (singlet, 3H); delta 2.90 (singlet4H), delta 2.70 (multiplet, 2H), delta 2.37 (multiplet, 2H), and delta1.79 (multiplet 4H).

A solution of methyl succinimidyl adipate (4.5 g, 17.5 mmol),chlorotrimethylsilane (11.1 ml, 87.5 mmol) and sodium iodide (13.1 g,87.5 mmol) in 10 ml of acetonitrile was heated at reflux for 12 hours.The mixture was then cooled to room temperature and diluted with ethylacetate. The reaction mixture was washed repeatedly with 5 percentaqueous sodium bisulfite until the organic solution was colorless. Thenit was washed with brine, dried over anhydrous magnesium sulfate,filtered and concentrated to provide 3.2 g (71 percent yield by weight)of adipic acid monosuccinimidyl ester as a white solid.

Proton NMR (in CDCl₃) delta 3.90 (singlet, 4H), delta 2.70 (multiplet,2H), delta 2.4 (multiplet, 2H), delta 1.80 (multiplet, 4H).

A mixture of adipic acid succinimidyl ester (1.00 g, 3.80 mmol) andthionyl chloride (5 ml) was heated at 40 degrees C. for 3 hours, thencooled to room temperature and concentrated in vacuo. The residue wasstirred several times with dry hexane, the oil was separated and driedin vacuo to provide 0.97 g (90 percent yield by weight) of succinimidyladipoyl chloride. This was dissolved in dry tetrahydrofuran to make a 5molar solution, which was used as such in the preparation of compoundssuitable for coupling to protein carriers.

Proton NMR (in CDCl₃) delta 3.00 (multiplet, 2H), delta 2.90 (singlet,4H), delta 2.70 (multiplet, 2H), delta 1.80 (multiplet 4H).

EXAMPLE 2 N-Hydroxysuccinimidyl glutaryl chloride (Linking Agent)

One mole of glutaric anhydride and one mole of N-hydroxysuccinimide weretreated in dichloromethane with 1.2 equivalents of triethylamine. Thisreaction was stirred for 40 minutes, acidified by the addition of 0.5Nhydrochloric acid (HCl) (aqueous), and then extracted with ethyl acetateto form mono N-hydroxysuccinimide glutarate. This glutarate was thencombined with 5 moles of thionyl chloride and stirred at roomtemperature for three hours. The resulting reaction mixture was dried toremove volatiles, and thereby form N-hydroxysuccinimidyl glutarylchloride.

EXAMPLE 3 2-Phenoxy-2-oxo-6-(aminomethyl)-1,2-oxaphosphorinane

One equivalent of phenylphosphorodichloridite was combined with twoequivalents of isopropanol and 2 equivalents of triethylamine, andreacted in diethylether for one hour at room temperature as described byTolkwith et al., J. Org. Chem., 23, 1682 (1958), to producediisopropylphenylphosphite in 49 percent yield. Boiling point=117-118degrees C. at 11 mm Hg.

Three equivalents of this phosphite were mixed with one equivalent of5-bromo-1-pentene and a catalytic amount of about 0.1 equivalent ofsodium iodide, and heated at 170-185 degrees C. for three hours toproduce phenyl isopropyl 4-pentenylphosphonate in 80 percent yield afterflash colum chromatography on silica. Boiling point=148-150 degrees C.at 1.2 mm Hg.

One equivalent of this phosphonate and 1.8 equivalents of iodine werestirred in chloroform at 10 degrees C. for three-four days as describedby Zhao et al., J. Org. Chem., 50, 2136 (1985) to produce2-phenoxy-2-oxo-6-iodomethyl-1,2-oxaphosphorinane in 61 percent yield.Melting point=127.5-128 degrees C.

A suspension containing two equivalents of sodium azide, one equivalentof the above-prepared iodonated oxaphosphorinane and a catalytic amountof about 0.1 equivalent of tetrabutylammonium bromide in benzene/DMF(1:1) was heated to 60-80 degrees C. for 16 hours to produce2-phenoxy-2-oxo-6-(azidomethyl)-1,2-oxaphosphorinane in 96 percent yieldas a white solid.

A solution of the above azide was prepared in ethanol containing 40percent by weight of 10 percent palladium on carbon and shaken under 40pounds per square inch (psi) of hydrogen gas for 20-24 hours at roomtemperature to produce the corresponding amine that was isolated as aviscous oil in a yield of 84 percent. A solution of the amine was thentreated in ether with an excess of pyridinium bisulfate to precipitatethe amine derivative by forming a bisulfate salt[2-phenoxy-2-oxo-6-aminomethyl-1,2-oxaphosphorinane bisulfate; CompoundA bisulfate].

The salt so obtained was neutralized with 5 percent aqueous sodiumbicarbonate, extracted into ethyl acetate, and recovered by evaporationof the solvent. The resulting dried amine (Compound A) was dissolved indichloromethane and treated first with one equivalent of theN-hydroxysuccinimide (NHS) ester of glutaryl chloride and then with 1.2equivalents of triethylamine to form the NHS-activated glutaramide.

The synthesis of this compound was itself stereospecific, yielding onlyone diastereomer (one pair of enantiomers) as revealed by a single 31PNMR resonance at 27.2 ppm relative to 85% H₃ PO₄. Confirmation that thephenoxy and aminomethyl substituents were in a 1,3-trans orientation(axial-equatorial) was obtained from the X-ray crystal structure of thepredecessor iodomethyl derivative.

Protein conjugates with the cyclic phosphonate of formula II wherein Rwas the described succinimidyl glutaryl chloride (Compound X) wereprepared by the addition of 0.250 ml of a solution of the phosphonate inDMF [5 milligrams (mg)/ml]to 0.75 ml of a solution of protein (KLH orBSA, 3.33 mg/ml) in sodium phosphate buffer (pH 7.2, 0.2M) and stirringgently for one hour at 22 degrees C.

The amine described above (Compound A) was also dissolved indichloromethane and treated with acetic anhydride and triethylamine toform an acetamide derivative (formula II, where R is methyl) afterpurification by flash column chromatography on silica in a yield of 28percent as a glass. This derivative is utilized as an inhibitor.

EXAMPLE 4 Phenyl-6-acetamido-5-trimethylsilyloxyhexanoate

One equivalent of mono methyl glutarate (Aldrich Chemical Corp.,Milwaukee, Wis.) was combined with 1.5 equivalents of thionyl chlorideand stirred at room temperature for one hour to form methylglutarylchloride. Volatile products were removed in vacuo from theresulting compound that was then treated with Cu^(I) CN in acetonitrileat 80 degrees C. for 15 hours as described by Hunig et al., Angew. Chem.Int. Ed. (Eng), 21, 36-49 (1982) (ref. 5), to form an acyl cyanide.

4.7 Grams of the acyl cyanide were shaken in 45 mililiters (ml) ofacetic acid and 4.5 ml of acetic anhydride containing 5 percentpalladium on carbon (1.12 gm) while under 40 psi of hydrogen gas to formmethyl 6-acetamido-5-oxohexanoate as a crystalline solid in 30 percentyield from mono methyl glutarate. The resulting hexanoate was stirredunder reflux in 95 percent ethanol containing 1.5 equivalents ofpotassium hydroxide for 5 minutes to form 6-acetamido-5-oxohexanoicacid. The resulting hexanoic acid was combined with 1.5 equivalents ofphenol and stirred with dicyclohexylcarbodiimide (DCC) anddimethylaminopyridine in dichloromethane as described by Hassner et al,Tet. Lett., 1978, 4475-4478 (1978) to form phenyl6-acetamido-5-oxohexanoate in 26 percent yield as a crystalline solid.The resulting hexanoate was then combined in methanol with threeequivalents of sodium cyanoborohydride, and while stirring, the pH valueof about 3 was maintained by the addition of a solution of methanolicHCl to form the corresponding alcohol. The alcohol (phenyl6-acetamido-5-hydroxyhexanoate, formula I where R is methyl) was thenconverted to the corresponding phenyl6-acetamido-5-trimethysilyloxyhexanoate, in 96 percent yield as a glassysolid from the above ketone, as described by Sweeley et al., J. Am.Chem. Soc., 85, 2497 (1963) for storage by reaction withtrimethylsilylchloride and bis(trimethylsilyl)amine in pyridine for 5minutes, and was reconverted to the alcohol for use as a substrateligand by reaction in 0.1 molar citric acid in methanol for 30 secondsat room temperature immediately prior to use.

EXAMPLE 5 Preparation of Monoclonal Receptors

The foregoing KLH conjugates were used to immunize mice (129GlX⁺strain), and monoclonal antibodies were obtained as described by Nimanet al., Proc. Natl. Acad. Sci. USA, 77, 4524 (1980) and Niman et al., inMonoclonal Antibodies and T-Cell Products, ed., Katz, D. H., 23-51 (CRCPress, Boca Raton, Fla. 1982).

The lymphocytes employed to form the hybridomas of the present inventioncan be derived from any mammal, such as a primate, rodent (e.g., mouseor rat), rabbit, guinea pig, cow, dog, sheep, pig or the like. Asappropriate, the host can be sensitized by injection of the immunogen,in this instance a haptenic analog-ligand, followed by a boosterinjection, and then isolation of the spleen.

It is preferred that the myeloma cell line be from the same species asthe lymphocytes. Therefore, fused hybrids such as mouse-mouse hybrids[Shulman et al., Nature, 276, 269 (1978)] or rat-rat hybrids [Galfre etal., Nature, 277, 131 (1979)] are typically utilized. However, somerat-mouse hybrids have also been successfully used in forming hybridomas[Goding, "Production of Monoclonal Antibodies by Cell Fusion," inAntibody as a Tool, Marchalonis et al. eds., John Wiley & Sons Ltd., p.273 (1982)]. Suitable myeloma lines for use in the present inventioninclude MPC-11 (ATCC CRL 167), P3×63-Ag8.653 (ATCC CRL 1580), Sp2/O-Ag14(ATCC CRL 1581), P3×63 Ag8U.1 (ATCC CRL 1597), Y3-Ag1.2.3. (deposited atCollection Nationale de Cultures de Microorganisms, Paris, France,number I-078) and P3×63Ag8 (ATCC TIB 9). The non-secreting murinemyeloma line Sp2/0 or Sp2 /O-Ag14 is preferred for use in the presentinvention.

The hybridoma cells that are ultimately produced can be culturedfollowing usual in vitro tissue culture techniques for such cells as arewell known. More preferably, the hybridoma cells are cultured in animalsusing similarly well known techniques with the monoclonal receptorsbeing obtained from the ascites fluid so generated. The animals used forgeneration of the ascites fluid were female 129GlX⁺ mice bred in themouse colony of the Scripps Clinic and Research Foundation, La Jolla,Calif., however, when animals other than mice are used for preparationof the hybridomas, mice or that animal type can be used for theproduction of ascites fluid.

In particular, exemplary monoclonal receptors were produced by thestandard hybridoma technology of Kohler et al., Nature, 256, 495 (1975).For the preparation of the exemplary monoclonal receptor designated24B11, the foregoing immunization protocol was modified as follows.Female 129GlX⁺ mice were immunized by intraperitoneal injection with aninoculum of conjugate containing 60 micrograms (ug) of Compound X boundto 125 micrograms of KLH in 500 microliters (ul) of emulsion comprisedof a 1:1 mixture of phosphate buffered saline (PBS) pH 7.4 and completeFreund's adjuvant. Two weeks later, the mice were again injected in alike manner with an innoculum containing one half the amount of originalconjugate in 500 microliters of a solution of a 1:1 mixture of PBS (pH7.4) and 10 mg/ml alum. After an additional four weeks, the mice wereimmunized intravenously with one and one-half times the amount oforiginal conjugate in 500 microliters of PBS (pH 7.4). The spleens wereremoved from the mice 5 days later, and the spleens were fused tomyeloma cells.

The spleens cells were pooled and a single cell suspension was made.Nucleated spleen cells (1.4×10⁸) were then fused with 3×10⁷ Sp2/0non-secreting myeloma cells in the presence of a cell fusion promoter(polyethylene glycol 2000). The hybridoma that produced a particularmonoclonal antibody was selected by seeding the spleen cells in 96-wellplates and by growth in Dulbecco's modified Eagle medium (DMEM)containing 4500 mg/liter glucose (10 percent), 10 percent fetal calfserum (FCS), hypoxanthine, aminopterin and thymidine (i.e., HAT medium)which does not support growth of the unfused myeloma cells.

After two to three weeks, the supernatant above the cell clone in eachwell was sampled and tested by ELISA (enzyme linked immunosorbent assayas described hereafter) for the presence of antibodies against theimmunizing analog-ligand. Positive wells were cloned twice by limitingdilution. Those clones that continued to produce analog-ligand-specificantibody after two clonings were expanded to produce larger volumes ofsupernatant fluid.

Twenty-four secreted monoclonal antibodies were further screened forhydrolytic activity (25 mM phosphate buffer at a pH 7.0, 25 degrees C.)by monitoring substrate depletion using high performance liquidchromatography (HPLC). One hybridoma and its monoclonal antibody, 24B11,were chosen for further study of catalytic activity.

The hybridoma and the monoclonal receptors produced therefrom anddescribed herein are identified by the designation 24B11, the particularmaterial referred to being apparent from the context. Hybridoma 24B11was deposited on Aug. 4, 1987 at the American Type Culture Collection,Rockville, Md. and were given the ATCC accession number HB9488.

The present deposits were made in compliance with the Budapest Treatyrequirements that the duration of the deposits should be for 30 yearsfrom the date of deposit or for 5 years after the last request for thedeposit at the depository or for the enforceable life of a U.S. patentthat matures from this application, whichever is longer. The hybridomaswill be replenished should they become non-viable at the depository.

A monoclonal receptor of the present invention can be produced byintroducing, as by injection, the hybridoma into the peritoneal cavityof a mammal such as a mouse. Preferably, as already noted, syngenic orsemi-syngenic mammals are used, as in U.S. Pat. No. 4,361,549, thedisclosure of which is incorporated herein by reference. Theintroduction of the hybridoma causes formation of antibody-producinghybridomas after a suitable period of growth, e.g. 1-2 weeks, andresults in a high concentration of the receptor being produced that canbe recovered from the bloodstream and peritoneal exudate (ascites) ofthe host mouse. Although the host mice also have normal antibodies intheir blood and ascites, the concentration of normal antibodies istypically only about five percent that of the monoclonal receptorconcentration.

The monoclonal receptor present in the hybridoma supernatant can be usedwithout purification or the receptor can be recovered from the ascitesor serum of the mouse using standard techniques such as affinitychromatography using AD 169-infected cells bound to an immunosorbantsuch Sepharose 6B or 4B (Pharmacia Fine Chemicals, Piscataway, N.J.),followed by elution from the immunosorbant using an acidic buffer suchas glycine hydrochloride at a pH value of about 2.5.

In the present studies, IgG fractions were obtained from mouse ascitesby precipitation with 45 percent saturated ammonium sulfate followed bychromatography on DEAE-Sephacel with sodium chloride elution. Thefraction that was eluted with 100 mM salt was dialyzed and concentrated.Stock solutions of antibody at 20 mg/ml were prepared in Tris-HCl (50mM, pH 6.5). Protein concentrations were determined by the Lowry method.[J. Biol. Chem., 193, 265 (1951)].

EXAMPLE 6 Enzyme-linked Immunosorbent Assay (ELISA)

The binding of ligands and the effect of chemical modification wereassayed by ELISA with antibody at a fixed concentration in the range ofits titer, and varying the reagent or ligand concentration. Inhibitionis reported if the titer is reduced 50 percent at less than a 1000:1ratio of reagent to hapten.

Assays were performed in flat-bottom polyvinyl microtiter plates(Dynatech, Alexandria, Va.). The wells were coated with a solutioncomprising analog-ligand bound to BSA as the antigen ligand in phosphatebuffered saline (PBS) using 50 microliters of solution per well. Theligand was coated at 1 microgram per milliliter. The plates were thenincubated overnight at 37 degrees C. in a dry oven. The dried plateswere stored at 4 degrees C. until use. Prior to the ELISA assay, driedplates were rehydrated by two washes of 2 minutes each with 10millimolar (mM) PBS, pH 7.4, containing 0.1 percent polyoxalkylene (20)sorbitan monolaurate (Tween 20) and 0.02 percent Thimerosal (sodiumethylmercurithiosalicylate), (Sigma, St. Louis, Mo.).

In order to reduce non-specific binding, hybridoma supernatants werediluted 1:2 in washing buffer containing 0.1 percent BSA as diluent.Fifty microliters of diluted hybridoma supernatants were thereafteradded to each well and incubated for 1 hour at 4 degrees C. on agyroshaker to contact the monoclonal antibody-containing supernatantwith the bound analog-ligand. Following two washes of 2 minutes each, 50microliters of peroxidase-labeled goat anti-mouse IgG+IgM (Tago,Burlingame, Calif.), diluted 1:1000, were added to each well, and thereaction mixture was incubated at 4 degrees C. for 1 hour to bind thelabeled antibody to bound monoclonal antibody.

The substrate used to assay bound peroxidase activity was prepared justprior to use and consisted of 400 microgram/ml o-phenylenediamine(Sigma, St. Louis, Mo.) in 80 mM citrate-phosphate buffer, pH 6.0,containing 0.12 percent H₂ O₂. After two final washes, 50 microliters ofsubstrate solution were added to each well and color was allowed todevelop for 15 minutes in the dark. Color development was stopped byadding 25 microliters of 4 molar (M) H₂ SO₄ to each well and the opticaldensity at 492 nanometers (nm) was measured with a Multiskan ELISA platereader. Polyclonal antibodies raised to the above analog-ligand wereobserved to immunoreact (bind) to the analog-ligand.

EXAMPLE 7 Comparative Kinetic Studies

first composition was prepared that contained about 0.5 mM of a reactantligand of formula I where R is methyl (phenyl6-acetamido-5-hydroxyhexanoate) as substrate and 5 uM of a non-specificmonoclonal antibody designated 24E12 in aqueous 50 mM phosphate asaqueous medium. This first composition was utilized as a control. Asecond composition was prepared that contained the same amount ofreactant ligand substrate and 5 uM receptor 24B11 as a lactone synthase.A third composition was prepared identical to the second composition butfurther containing 20 uM of2-phenoxy-2-oxo-6-(acetamidomethyl)-1,2-oxaphosphorinane (analog-ligandof formula II where R is methyl) as inhibitor.

The three compositions were maintained at room temperature and aliquotsfrom each were taken at intervals to ascertain the concentration ofsubstrate ligand remaining in each composition. Assays were carried outusing HPLC (Hitachi) with a solvent containing 20 percent acetonitrile,79.9 percent water and 0.1 percent trifluoroacetic acid, using a flowrate of 1.5 ml per minute. The detector was set at 225 nm. A C18 reversephase column using Vidac 218TP54 as the solid phase was used.

As can be seen from examination of the data of FIG. 2, the concentrationof substrate ligand in above Compositions 1 (control) and 3 (substrateplus inhibitor) decreased at substantially identical rates. The decreasein substrate ligand in Composition 2 that contained the lactone synthaseand no inhibitor decreased much more rapidly until about one-half of thesubstrate was consumed, and thereafter decreased at a rate similar tothat of Compositions 1 and 3. (The background decreases in substrate inCompositions 1, 2 and 3 are thought to be due to non-specific hydrolysisof the phenyl ester.)

Since the substrate was present at a one hundred-fold excess over thereceptor, and since at least one-half of the substrate was consumedrelatively rapidly, the results shown in FIG. 2 indicate that thereaction involving the receptor was greater than stoichiometric and thattrue catalysis with turn over of the receptor catalyst with synthaseactivity was observed.

A likely explanation for the relatively rapid decrease to about one-halfthe initial concentration of substrate followed by the backgroundsubstrate decrease is that the receptor synthase binds to the ligandsubstrate and catalyzes the lactone-forming reaction stereospecifically.Thus, the ligand has an asymmetric carbon atom at the 5-position wherethe hydroxyl, hydrogen and methylene acetamido groups are bonded to thechain; i.e., the alpha-carbon of the alcohol portion of the molecule. Asa consequence, the ligand substrate exists as an enantiomeric pair. Asimilar enantiomeric pair exists for the analog-ligand utilized toinduce production of the receptor lactone synthase.

If the receptor lactone synthase 24B11 secreted by hybridoma 24B11 boundonly one of the enantiomers of both the immunizing analog-ligand andsubstrate ligand, but not to the other enantiomer of either, it would beexpected to catalyze lactone formation only by the substrate ligand towhich it bound. Since each enantiomer constitutes one-half of theconcentration of the enantiomeric pair, lactone formation of the boundenantiomer would be expected to be stereospecific and utilize onlyone-half of the admixed substrate ligand as is observed.

A schematic representation of the lactone synthase-catalyzed reactionillustrated for one enantiomer of the substrate ligand and oneimmunizing analog-ligand that is an analog to the lactone-formingtransition state is shown in FIG. 1, using usual stereochemicaldepictions for chemical bonds. As is seen from that Figure, oneenantiomer of the substate ligand (shown on the left) can form atransition state (shown in the brackets in the upper center of theFigure) that has a stereochemistry substantially identical to that ofthe analog-ligand (shown in brackets in the lower center). The lactoneproduct (shown on the right of the Figure) is a single stereoisomer thatretains the configuration of the ligand enantiomer that was bound and isshown on the left.

EXAMPLE 8 Reaction Kinetics

Further kinetic determinations were made using the substrate ligand,monoclonal receptor lactone synthase and inhibitor of Example 7. Here,the receptor was present at a concentration of 2 uM from a Lowry assayand a presumed molecular weight of 150,000 daltons for an IgG antibody,along with 20-100 uM of the reactant ligand (substrate ligand) and 0.25uM or 0.50 uM inhibitor, where used.

Phenol release from the substrate ligand was determinedspectrophotometrically at 271 nm, and initial rates as a function ofsubstrate concentration followed Michaelis-Menten kinetics consistentwith the reaction sequence shown in the upper portion of FIG. 3. Thelower portion of FIG. 3 shows Lineweaver-Burk plots for the inverse ofinitial rates (1/V) versus the inverse of the substrate ligandconcentration (1/[S]). Relevant kinetic parameters in the presence andabsence of the catalytic lactone synthase molecule are shown in Table 1,below, and indicate a rate acceleration of 167-fold.

                  TABLE 1                                                         ______________________________________                                        Kinetic Parameters                                                             (uM)K.sub.m *                                                                       (uM)K.sub.i                                                                            (uM/min)V.sub.max                                                                       (l/min)k.sub.cat                                                                      (l/min)k.sub.uncat                                                                   ##STR7##                             ______________________________________                                        76    0.25     0.99      0.50    0.003  167                                   ______________________________________                                         *In the absence of binding by the unreactive enantiomer of the substrate      ligand, K.sub.m is about 38 uM for the reactive enantiomer of the             substrate ligand.                                                        

Control reactions were run to confirm that the catalytic activityobserved was a property of the lactone synthase, 24B11, and not acontaminating esterase. It was found that neither the hydrolyticallymore labile coumarin ester of the substrate ligand nor coumaryl5-hydroxypentanoate, which lacks the acetamidomethyl group of thesubstrate that can act as a recognition element, were substrates forcatalysis. Another receptor designated 24E12 bound to the immunizinganalog-ligand but did not catalyze liberation of phenol from thesubstrate ligand.

The reaction of the lactone synthase 24B11 with the substrate waslineary competitively inhibited by the addition of the N-acetylderivative of the analog-ligand as can be seen from the before-mentionedLineweaver-Burk plots of FIG. 3. The rate constant for that inhibition,K_(i), was found to be 0.25 uM.

EXAMPLE 9 Lactone Formation is Stereospecific

The data in Example 7 indicated that only about one-half (about 50percent) of the added substrate was consumed relatively rapidly torelease phenol, whereas the remaining phenol was liberated at a ratesimilar to that observed in the absence of the receptor synthasemolecule. A second aliquot of substrate ligand was admixed with thereaction mixture after the first, relatively fast portion of thereaction was complete. That second admixture again resulted in arelatively rapid about 50 percent depletion of reactant ligandsubstrate, with the second depletion occurring at about the same rate asthe first depletion. These results are shown in FIG. 4, wherein thefirst reactant ligand admixture is shown at point A, and the second isshown at point B.

Introduction of further receptor synthase 24B11 was without effect.Injection of an aliquot of pheonl (0.5 equivalents of the reactantligand) provided the expected absorbance increase.

These results indicate the there was not inhibition of the reaction byone of its products, phenol, and also that phenol absorption was notsomehow being masked. Furthermore, HPLC analysis of the reaction mediumjust after the relatively fast first phase was completed yielded about50 percent of the reactant ligand.

Taken together, these results were consistent with a stereospecificreceptor synthase-catalyzed reaction of one enantiomer of the reactantligand substrate. There was no evidence for cyclization of the otherenantiomer being catalyzed by the receptor synthase over the observedtime course of about 30 minutes.

EXAMPLE 10 Confirmation of Stereospecific Synthesis

To confirm that the lactone-forming reaction was catalyzedstereospecifically, the lactone product enantiomers were independentlysynthesized by treatment of 6-iodomethylvalerolactone [Shamma et al., J.Am. Chem. Soc., 76:2315 (1954)] with sodium azide, followed by reductionin the presence of acetic anhydride. Extraction and chromatographicpurification of the lactone permitted examination of enantiomeric purityby ¹ H NMR in the presence of a chiral lanthanide shift reagent,tris-[3-(heptafluoropropylhydroxymethylene)-d-camphorato]-europium(III), Eu(hfbc)₃, utilizing the acetamido function to bind the reagent.Sullivan, Top. Steroreochem., 10:287 (1978).

Clear separation of each of the three single ¹ H NMR resonances for theprotons of the CH₃ CONH-substituent and of the sidechain CH₂ of theproduct lactone into two proton signals was obtained for both thereceptor synthase-generated and chemically-synthesized lactone products.A portion of each spectrum is shown in FIG. 5.

The equivalence of peak areas for the synthetic sample as expected for aracemic modification validates the analytical method and indicates thatthe observed enantiomer excess (% major peak - % minor peak) generatedby the receptor synthase-catalyzed cyclization was 66±4 percent.

That latter percentage can be corrected for the competing spontaneouscyclization reaction of the substrate under the study conditions using acomputer simulation of the equation shown in the upper portion of FIG. 3by employing the rate constants of Table 1. That computer simulationpredicted that about 86 percent of the minor peak arose from theuncatalyzed, spontaneous reaction. It is noted that it is not necessaryto introduce explicitly a term for the spontaneous hydrolysis of thelactone product since both enantiomers decay at identical rates, thusmaintaining their relative ratio.

This analysis indicates that the stereospecificty of the receptorsynthase-catalyzed lactone formation of the reactant ligand substratefavored one enantiomer in 94±8 percent excess, given the limits of thedeterminations. Thus, the work described herein documents the firstantibody combining site-catalyzed bond-forming, as compared tohydrolytic, reaction; a cyclization reaction. That work moresignificantly also exemplifes the first demonstration of sterochemicalcontrol of a reaction course, as is so typical of an enzyme, catalyzedby an antibody combining site-containing molecule.

EXAMPLE 11 Receptor-Mediated Separation of Stereoisomers

The previous description discussed several manners by which usefulreceptor molecules could be used to separate stereoisomers from eachother. This Example provides a step-by-step preparation of an affinitysorbant, affinity column and the separation of the most similar ofstereoisomers, enantiomers.

Sepharose 4B-CL (Pharmacia Fine Chemicals, Piscataway, N.J.), across-linked agarose, is utilized as a water-insoluble matrix and isadmixed with 2M Na₂ CO₃. The admixture is packed by centrifugation andthe supernatant liquid is then discarded. Two volumes of 2M Na₂ CO₃ areadded to the packed Sepharose and that admixture is resuspended in acapped bottle.

Cyanogen bromide (CNBr) from a stock solution at a concentration of onegram per milliliter (g/ml) in water-free acetonitrile is added to theSepharose 4B-CL suspension in an amount of 0.2 volumes of CNBr solutionper volume of Sepharose 4B-CL (at 200 mg per ml Sepharose 4B-CL). Thebottle is capped and the contents are mixed vigorously for a time periodof 2-3 minutes at room temperature.

The suspension is poured onto a sintered glass funnel and washed undersuction with: (1) ten volumes of 0.1M NaHCO₃, pH 9.5; (2) 10 volumes ofdistilled water; and (3) 10 volumes of coupling buffer [(PBS); 0.02Mphosphate, 0.15M NaCl, pH 7.5] to form CNBr-activated Sepharose 4B-CL.The activated Sepharose is removed from the filter and admixed with asolution containing monoclonal antibody 24B11 at 5 mg of antibody peroriginal packed volume of Sepharose 4B-CL in coupling buffer to couplethe antibody to the activated Sepharose. The coupling reaction ismaintained for 2-4 hours at room temperature with tumbling mixing.

The antibody-coupled Sepharose is thereafer admixed with 1-2 volumes of1M ethanolamine, pH 8.0, to block any remaining reactive groups. Theresulting admixture is maintained at room temperature for a time periodof 2-4 hours. The resulting blocked 24B11 antibody-bound Sepharosewater-insoluble affinity sorbant is thereafter packed in a column andthoroughly washed with the buffer utilized for separating thestereoisomers (below) until no further protein elutes from the columneluate.

The above procedure is that reported in Antibody As A Tool, Marchalonisand Warr eds., John Wiley & Sons, New York (1982), pages 89-91 whereinroutine coupling efficiencies in excess of 80 percent are reported whenoffering IgG at a ratio of 5 mg/ml of Sepharose.

A column containing the blocked 24B11 antibody-bound Sepharose 4B-CL inan amount of 2 ml, based upon the original packed, volume, is preparedusing 25 mM phosphate buffer, pH 7.0, as the eluting buffer, and iswashed as discussed before. One milliliter of a solution containing theenantiomeric analog-ligand of formula II where R is methyl at aconcentration of 50 uM is added to the top of the column, and thereaftereluted therefrom. The original analog-ligand-containing solution is notoptically active, whereas the eluate from the column is opticallyactive, thereby demonstrating the separation of the enantiomericstereoisomers.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from true spirit and scope of the invention.

What is claimed is:
 1. A method of separating one of a pair ofenantiomers from the other comprising admixing a mixture of enantiomerswith a monoclonal receptor molecule containing an antibody combiningsite that immunoreacts with substantially only one of said enantiomersin an aqueuos medium to form an admixture, said receptor moleculebinding to:(a) substantially only one of an enantiomeric reactant ligandpair structurally capable of forming an enantiomeric amide or esterproduct, said reactant ligand containing a carbonyl group carbon atomand an amine or alcohol group structurally capable of forming thepreselected enantiomeric carboxylic acid amide or ester product; and (b)a ligand structurally analogous to one enantiomer of a transition stateleading to said preselected amide or ester product, the enantiomer ofsaid analog-ligand that is bound by said combining site having atetrahedrally bonded phosphorus atom located at the position occupied bythe carbon atom of the carbonyl group of the carboxylic acid amide orester product said tetrahedrally bonded phosphorus atom being bondeddirectly to:(i) the alpha-carbon atom of the acid portion of saidanalog-ligand by a single bond; (ii) a first oxygen atom that is doublybonded to said phosphorus; (iii) a second oxygen atom that is singlybonded to said phosphorus atom, and is singly bonded to a radicalselected from the group consisting of hydrogen, C₁ -C₆ lower alkyl,benzyl and phenyl; and (iv) a third oxygen atom or a nitrogen atom thatis singly bonded to said phosphorus atom, and is also singly bonded tothe alpha-carbon atom of the amine or alcohol portion of saidanalog-ligand; maintaining said admixture for a time period sufficientfor said receptor to bind to one of said enantiomers and to form animmunoreactant with said receptor within said admixture; and separatingsaid immunoreactant from the remaining admixture.
 2. The method of claim1 wherein said receptor is linked to a solid phase matrix.
 3. The methodof claim 1 wherein said monoclonal receptor is an intact antibody. 4.The method of claim 3 wherein said intact antibody is secreted byhybridoma 24B11.
 5. The method of claim 1 including the further step ofseparating the bound enantiomer from the receptor of the immunoreactant.